Methods of modulating cold sensory perception via a cold- and menthol-sensitive receptor (CMR1)

ABSTRACT

The present invention relates to regulation of cold sensation and pain. More particularly, the present invention is directed to nucleic acids encoding a member of the transient regulatory protein family, CMR1, which is involved in modulation of the perception of cold sensations and pain. The invention further relates to methods for identifying and using agents that modulate cold responses and pain responses stimulated by cold via modulation of CMR1 and CMR1-related signal transduction.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/340,443, filed Dec. 29, 2011, now U.S. Pat. No. 8,470,545, whichis a divisional application of U.S. Ser. No. 12/905,001, filed Oct. 14,2010, now U.S. Pat. No. 8,361,733, which is a divisional application ofU.S. Ser. No. 12/032,485, now U.S. Pat. No. 7,838,253, filed Feb. 15,2008, which is a divisional application of U.S. Ser. No. 10/352,724, nowU.S. Pat. No. 7,371,841, filed Jan. 27, 2003, which claims priority toU.S. Ser. No. 60/351,974, filed Jan. 25, 2002, and U.S. Ser. No.60/355,037, filed Feb. 7, 2002, which are herein incorporated byreference in their entireties.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under grant numberGM44298 awarded by the National Institutes of Health. The Government hascertain rights in this invention.

REFERENCE TO A “SEQUENCE LISTING, ” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The sequence listing written file -1232-7.TXT, created on Jun. 26, 2013,32,768 bytes, machine format IBM-PC, MS-Windows operating system, ishereby incorporated by reference in its entirety for all purpose.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing as a text file named“-1232-6.txt” created Dec. 29, 2011 and containing 29,778 bytes. Thematerial contained in this text file is incorporated by reference in itsentirety for all purposes.

FIELD OF THE INVENTION

The current invention relates to regulation of cold sensation. Moreparticularly, the present invention is directed to nucleic acidsencoding a member of the transient receptor potential (TRP) ion channelfamily, cold- and menthol-sensitive receptor (CMR1), which is involvedin detecting cold stimuli. The invention further relates to methods foridentifying and using agents, including small organic molecules,antibodies, peptides, nucleic acids, antisense nucleic acids, andribozymes, that modulate cold sensation via modulation of CMR1; as wellas to the use of expression profiles and compositions in diagnosis andtherapy related to cold sensation.

BACKGROUND OF THE INVENTION

The somatosensory system can detect changes in ambient temperature overa remarkably wide range, enabling us to discriminate among thermalstimuli of an innocuous (cool or warm) or noxious (cold or heat)quality. This process is initiated when a thermal stimulus excites theperipheral terminals of primary sensory neurons from dorsal root ortrigeminal ganglia, which innervate regions of the trunk and head,respectively. These neurons convert thermal stimuli into electrochemicalsignals (i.e. action potentials) and relay information to integrativecenters in the spinal cord and brain (Fields, Pain (1987); Julius &Basbaum, Nature 413:203-10 (2001)). Noxious (painful) heat is detectedby primary sensory neurons that respond with a moderate thermalthreshold of ˜43° C. or with a high threshold of ˜52° C. (Raja et al.,in Textbook of Pain, pages 11-57 (Wall & Melzack, eds., 1999); Nagy &Rang, Neuroscience 88:995-7 (1999)). Insights into the molecularmechanisms of heat sensation have come from the cloning andcharacterization of the vanilloid receptor (VR1), an excitatory ionchannel on sensory neurons that is activated by capsaicin, the mainpungent ingredient in “hot” chili peppers and is also activated bynoxious heat at temperatures >43° C. (Caterina et al., Nature 389:816-24(1997)). Indeed, electrophysiological, anatomical, and genetic studiessupport this hypothesis and show that VR1 is essential for thedevelopment of thermal hypersensitivity following tissue injury(Caterina & Julius, Annu. Rev. Neurosci. 24:487-517 (2001)). A relatedion channel, VRL-1, does not respond to capsaicin, but is activated bytemperatures >50° C., suggesting that it contributes to heat sensitivityof high threshold neurons (Caterina et al., Nature 398:436-41 (1999)).Both VR1 and VRL-1 belong to the transient receptor potential (TRP) ionchannel family.

In contrast to the understanding of noxious heat sensation, little isknown about how we detect cold. Recordings from cutaneous sensory nervesin the cat suggest that noxious cold (<15° C.) is detected primarily bytwo classes of unmyelinated C-fibers: those that also respond tohigh-threshold (noxious) mechanical and heat stimuli, and anotherpopulation that is activated by low-threshold (innocuous) mechanicalstimuli. Another class of C-fibers can be activated by moderate coolingof the skin to 25° C., but are mechanically insensitive (Bessou & Perl,J. Neurophysiol. 32:1025-43 (1969)). Interestingly, some fibers in thislatter class are also activated at temperatures >43° C., a phenomenonclassically described as a paradoxical response of cold fibers tonoxious heat (Campero et al., J. Physiol. 535:855-65 (2001); Dodt &Zotterman, Acta Physiol. Scand. 26:358-365 (1952)). Studies in rodentsshow that unmyelinated C-fibers as well as thinly myelinated M fibersare sensitive to noxious cold, but the percentage of such unitsresponding to cold ranges from ˜10% to 100%, depending on the stimulusintensity and species examined (Kress et al., J. Neurophysiol. 68:581-95(1992); Caterina et al., Science 288:306-13 (2000); Simone & Kajander,Neurosci. Lett. 213:53-6 (1996); Simone & Kajander, J. Neurophysiol.77:2049-60 (1997); Cain et al., J. Neurophysiol. 85:1561-74 (2001)).

This wide variability in the literature may reflect the fact thatthermal thresholds for cold-sensitive fibers are not as well defined asthey are for heat-sensitive units. Moreover, psychophysical thresholdsfor cold-evoked pain are not as precise as they are for heat and thusfiber types that transduce sensations of innocuous cool or noxious coldare not as firmly established. At the cellular level, calcium-imagingand patch-clamp studies of dissociated dorsal root ganglion (DRG)neurons have shown that cold (˜20° C.) promotes calcium influx, possiblythrough the direct opening of calcium-permeable ion channels on thesecells (Reid & Flonta, Nature 413:480 (2001); Suto & Gotoh, Neuroscience92:1131-5 (1999)). However, several other mechanisms have been proposedto explain cold-evoked membrane depolarization, including inhibition ofbackground K⁺ channels (Reid & Flonta, Neurosci. Lett. 297:171-4(20010), activation of Na⁺-selective degenerin channels (Askwith et al.,Proc. Natl. Acad. Sci. U.S.A. 98:6459-63 (2001)), inhibition of (Na⁺/K⁺)ATPases (Pierau et al., Brain Res. 73:156-60 (1974)), or differentialeffects of cold on voltage-gated Na⁺ and K⁺ conductances (Braun et al.,Pflugers Arch. 386:1-9 (1980)). Thus it is not clear whether coldexcites sensory neurons by activating a discrete “cold receptor,” or bymodulating a constellation of excitatory and inhibitory channels onthese cells.

Fifty years ago, Hensel & Zotterman (Acta Physiol. Scand. 24:27-34(1951)) showed that menthol potentiates responses of trigeminal fibersto cold by shifting their thermal activation thresholds to warmertemperatures. Moreover, they proposed that cooling compounds mediatetheir psychophysical effects by interacting with a protein in sensoryneurons that is specific to the process of cold transduction. Althoughrecent studies of sensory nerve fibers or dissociated DRG neurons inculture support this idea, no unifying cellular mechanism has beenproposed to explain menthol's action. For example, one model proposesthat menthol inhibits voltage-dependent Ca²⁺ channels (Swandulla et al.,Pflugers Arch. 409:52-9 (1987)), thereby decreasing activation ofCa²⁺-dependent K⁺ channels and prolonging depolarization ofcold-sensitive fibers (Schafer et al., J Gen Physiol. 88:757-76 (1986)).Another model predicts that menthol directly activates calcium-permeableion channels on these cells (Reid & Flonta, Nature 413:480 (2001);Okazawa et al., Neuroreport 11: 2151-5 (2000)). In any case, there iscurrently no direct pharmacological or biochemical evidence to supportthe existence of a bona fide menthol binding site on sensory neurons,nor is it clear whether menthol and cold act through the same molecularentity to depolarize these cells.

Cold/menthol receptor gene and related genes have been reported in theliterature under various different names. For example, McKemy et al.,Nature 416:52-58 (2002) refer to this gene as CMR1 and suggest the roleof this gene as a cold receptor and also suggest a possible general rulefor TRP channels in thermosensation. Also, Peier et al. (Cell 108(5):705-715 (2002) and Science 296:2046-9 (2002)) refer to a TRP channelthat they name to TRPM8, which is reported to be a distant relative ofVR1, that is activated by cold temperatures and by a cooling agent,menthol. Additionally, Tsavaler et al. and others of DendreonCorporation refer to a gene related to CMR1 by the names Trp-8 and SP1-4, and teach that this gene is upregulated in prostate cancer andother malignancies.

The current invention is based on the discovery that the molecular siteof menthol action is an excitatory ion channel expressed bysmall-diameter neurons in trigeminal and dorsal root ganglia.Remarkably, the cloned channel is also activated by cold (8 to 28° C.),demonstrating that menthol does, indeed, elicit a sensation of cool byserving as a chemical agonist of a thermally responsive receptor. Thiscold- and menthol-sensitive receptor (CMR1) exhibits the highestsimilarity to members of the so-called long TRP or TRPM channelsubfamily, making it a close molecular cousin of the heat-activatedchannels, VR1 and VRL-1. Thus, TRP channels are the primary moleculartranducers of thermal stimuli and pain related to thermal stimuli withinthe mammalian somatosensory system.

SUMMARY OF THE INVENTION

The present invention therefore provides nucleic acids encoding a cold-and menthol-sensitive receptor, CMR1, including variants and chimerasthereof. The invention further provides methods of using CMR1polynucleotide and polypeptide sequence to screen for compounds,including small organic molecules, antibodies, peptides, lipids, nucleicacids, antisense molecules, siRNA molecules, and ribozymes, to modulatecold/cool sensation. Such compounds can be used as flavoring or perfumeagents or as components of medicaments to provide a cool or coldsensation. Additionally, compounds that modulate CMR1 activity may beused to modulate pain perception, e.g., in the treatment of pain and forthe treatment of pain induced by cool or cold and/or menthol stimulus.

In one aspect of the invention, nucleic acids encoding CMR1 protein, areprovided. In another aspect, the present invention provides nucleicacids, such as probes, antisense oligonucleotides, and ribozymes, thathybridize to a gene encoding a CMR1 protein, e.g., a nucleic acidsequence set out in SEQ ID NO:2 or 4. Often, the nucleic acid encodes aprotein comprising at least about 95% or 98% identity of an amino acidsequence set out in SEQ ID NO:1 or 3. Often, the nucleic acid is asequence comprising at least about 95% or 98% identity of an amino acidsequence set out in SEQ ID NO:2 or 4. In another aspect, the inventionprovides expression vectors and host cells comprising CMR1-encodingnucleic acids. In another aspect, the present invention provides CMR1proteins and antibodies thereto. The CMR1 protein can be fused to aheterologous protein, i.e., to make a fusion protein.

Often, the CMR1 polypeptide, variant, chimera, or fragment thereof, isrecombinant and the cell that expressed the CMR1 polypeptide, variant,chimera, or fragment thereof is a non-neuronal cell. In someembodiments, the host cell is from a mammalian cell line, e.g., a 293 ora CHO cell line.

In another aspect, the invention provides a method for identifying acompound that modulates cold/cool sensation or pain, the methodcomprising the steps of: (i) contacting a cell comprising a CMR1polypeptide, variant, chimera, or fragment thereof, with the compound,wherein the CMR1 polypeptide, variant, chimera, or fragment thereof isencoded by a nucleic acid that hybridizes under stringent conditions toa nucleic acid comprising a CMR1 nucleotide sequence of SEQ ID NO:2 or4, and forms a cation channel; and (ii) determining the chemical orphenotypic effect of the compound upon the cell comprising the CMR1polypeptide, thereby identifying a compound that modulates coldsensation or pain.

In another aspect, the present invention provides a method foridentifying a compound that modulates cold sensation or pain, the methodcomprising the steps of: (i) contacting the compound with a CMR1polypeptide, variant, chimera, or fragment thereof, the CMR1polypeptide, variant, chimera, or fragment thereof is encoded by anucleic acid that hybridizes under stringent conditions to a nucleicacid comprising a CMR1 nucleotide sequence; (ii) determining thephysical effect of the compound upon the CMR1 polypeptide; and (iii)determining the chemical or phenotypic effect of the compound upon acell comprising an CMR1 polypeptide or fragment thereof, therebyidentifying a compound that modulates cold sensation or pain.

In one embodiment, the CMR1 polypeptide or fragment thereof is encodedby a nucleic acid that hybridizes under stringent conditions to anucleic acid comprising a sequence of SEQ ID NO:2 or 4. Often, thenucleic acid encodes a CMR1 polypeptide comprising at least about 85%,95%, or 98% identity to an amino acid sequence set out in SEQ ID NO:1 or3. Often, the nucleic acid comprises at least about 85%, 95%, or 98%identity to a nucleic acid sequence set out in SEQ ID NO:2 or 4.

In another embodiment, the nucleic acid encodes a CMR1 polypeptide thathas at least 60% identity to an amino acid sequence set out in SEQ IDNO:1 or 3.

In a further embodiment, the method comprises contacting the compoundwith a CMR1 polypeptide or fragment thereof is encoded by a nucleic acidthat hybridizes under moderately stringent conditions to a nucleic acidcomprising a sequence of SEQ ID NO:2 or 4. Often, the nucleic acid hasat least 60% identity to a nucleic acid sequence set out in SEQ ID NO:2or 4.

In one embodiment, the chemical or phenotypic effect is determined bymeasuring CMR1 expression, intracellular Ca²⁺ mobilization, or changesin membrane currents.

In another embodiment, the CMR1 polypeptide is encoded by a nucleic acidcomprising a sequence set forth in SEQ ID NO:2 or 4. In anotherembodiment, the CMR1 polypeptide comprises an amino acid sequence of SEQID NO:1 or 3.

In one aspect, the present invention provides a method of modulatingcold sensation or pain in a subject, the method comprising the step ofadministering to the subject a therapeutically effective amount of acompound identified using the methods described above. The compound canbe administered using a variety of routes and formulations (see, e.g.,Remington's Pharmaceutical Sciences, 17th ed., 1989)

In one embodiment, the subject is a human.

In one embodiment, the present invention provides method of modulatingcold sensation or pain in a subject, the method comprising the step ofadministering to the subject a therapeutically effective amount of anCMR1 polypeptide, the polypeptide encoded by a nucleic acid thathybridizes under stringent conditions to a nucleic acid comprising aCMR1 nucleotide sequence.

In another aspect, the present invention provides a method of modulatingcold sensation or pain in a subject, the method comprising the step ofadministering to the subject a therapeutically effective amount of anucleic acid encoding a CMR1 polypeptide or fragment thereof, whereinthe nucleic acid hybridizes under stringent conditions to a nucleic acidencoding a polypeptide comprising a CMR1 nucleotide sequence.

In another aspect, the invention provides a compound capable ofmodulating a cold receptor identified using the methods disclosedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-F. A subset of trigeminal neurons express an inwardly rectifyingCa 2+-permeable channel activated by menthol and cold. A, Responses ofdissociated trigeminal neurons to cold (7° C.), menthol (500 μM), andcapsaicin (1 μM) were assessed by calcium imaging. Arrowheads markmenthol responding cells that were insensitive to capsaicin. Relativecalcium concentrations are indicated by Fura-2 ratio. The percentage(±s.e.m.) of excitable (potassium-sensitive) cells responding to eachstimulus is indicated below. B, Electrophysiological responses oftrigeminal neurons to menthol (50 μM), cyclohexanol (100 mM), menthone(100 μM), or cold (16° C.) were measured in both inward and outwarddirections (V_(hold)=−60 and +80 mV, respectively). Increasingtemperature of perfusate (from RT to 30° C.) completely antagonizedcurrents evoked by 100 μM menthol (right). Perfusate temperatures areindicated below each current trace. C, Menthol (50 μM, gray)- and cold(16°, black)-evoked responses show strong outward rectification.Changing the pipette solution to Cs-gluconate did not shift the reversalpotential significantly (not shown). Time dependence of menthol-evokedwhole-cell currents was analyzed using 400 ms voltage ramps ranging from−120 to +70 mV in 10 mV steps. Inset shows baseline currents (black) andmenthol-evoked responses (gray) in nominally Ca²⁺ free bath solution atRT using a CsCl-filled recording electrode. D, Voltage ramps (−120 to+80 mV in 200 ms) were used to establish current-voltage relationshipsin different extracellular solutions. Recording electrodes were filledwith standard pipette solution. Replacement of extracellular NaCl (140mM) with equimolar KCl or NMDG shifted the reversal potential fromE_(rev(Na))=−5.11±3.11 mV (n=6) to E_(rev(K))=−4.45±2.01 mV (n=3) andE_(rev(NMDG))=−84.99±11.51 mV (n=7). Replacement of extracellular NaClwith 125 mM NMDG and 10 mM CaCl₂ shifted the reversal toE_(rev(CaNMDG))=−43.28±4.59 mV (n=6, P_(Ca)/P_(Na)=3.22). Change ofpipette solution from standard (140 KCl, 5 CsCl) to 140 CsCl innominally Ca 2+ free bath solution shifted the reversal from 0.50±0.64mV (n=5) to −3.56±1.90 mV (n=6, not shown). E, Concentration-responsecurve for menthol-evoked inward currents (Vhold=−60 mV) in trigeminalneurons. Membrane currents in each neuron were normalized to 1 mMmenthol at room temperature. Each point represents mean value (±s.e.m.)from six independent neurons. Data were fit to the Hill equation. F,Temperature-response curves (from 33° C. to 16° C.) were determined fortrigeminal neurons in the presence (gray) or absence (black) of 10 μMmenthol. Menthol potentiated the size of cold-evoked currents andshifted thermal thresholds from 27.1±0.5° C. to 29.6±0.3° C. (n=4).

FIG. 2A-B. Cooling compounds activate the cloned receptor. A, An oocyteexpressing the cloned receptor was exposed to consecutive applicationsof menthol (100 μM), menthone (500 μM), cyclohexanol (500 μM),eucalyptol (20 mM), camphor (1 mM), icilin (300 nM), and capsaicin (1μM). Resulting membrane currents were measured under voltage clamp at−60 mV. Bars denote the duration of agonist application. Chemicalstructures for menthol, eucalyptol, and icilin are shown below theirrespective responses. B, Concentration-response curves for icilin(boxes), menthol (circles), and eucalyptol (triangles). Responses werenormalized to those evoked by 500 μM menthol. Each point represents meanvalues (±s.e.m.) from 4 to 9 independent oocytes.

FIG. 3A-E. Electrophysiological properties of menthol-induced currentsin transfected HEK293 cells. A, Time dependence of menthol inducedwhole-cell currents were analyzed using 400 ms voltage step pulsesranging from −120 to +70 mV in 10 mV steps (top). Traces show currentresponse induced by menthol (50 μM) at RT in nominally Ca 2+ free bathsolution using a CsCl-filled recording electrode. Current-voltagerelationship (bottom) was obtained from the same pulse protocol using200 μM menthol (RT) by plotting the time-independent current componentas a function of membrane voltage. Menthol currents reversed at−4.49±1.07 mV (±s.e.m., n=4) and show strong outward rectification. Asimilar current-voltage relationship was obtained for 2 μM icilin from a200 ms voltage ramp (−120 to +80 mV). B, 200 ms voltage ramps rangingfrom −120 to +80 mV were used to record current-voltage curves indifferent extracellular solutions. Recording electrodes were filled withstandard pipette solution. Replacement of extracellular NaCl (140 mM)with equimolar KCl or NMDG shifted the reversal potential fromE_(rev(Na))=−3.79±2.36 mV (n=13) to E_(rev(K))=0.82±1.02 mV (n=8) andE_(rev(NMDG))=−77.22±5.90 mV (n=11), respectively (P_(K)/P_(Na)=1.20,P_(NMDG)/P_(Na)=0.06). Replacement of extracellular NaCl with 125 mMNMDG and 10 mM CaCl₂ shifted the reversal to E_(rev(CaNMDG))=−35.16±8.00mV (n=9; PCa/PNa=3.34). Change of pipette solution from standard (140KCl, 5 CsCl) to 140 CsCl in nom. Ca²⁺ free bath solution shifted thereversal from −2.27±1.02 mV (n=3) to −4.49±1.07 mV (n=4,P_(Cs)/P_(K)=1.14, not shown). C, Responses evoked by consecutivementhol applications (50 mM, standard pipette solution) arecharacterized by a decrease of 53.9±1.7% (n=3) in peak current levelbetween first and second application in the presence of 2.5 mMextracellular Ca 2+ (black bars) and 9.1±7.0% in nominally Ca 2+ free(gray bar) bath solution (t-test; p=0.03; n=3). Icilin-evoked currentsdepend on extracellular Ca 2+ and desensitized by 78.8±17% (n=3) 20 safter application (relative to peak currents). D, Single channel traceswere recorded from transfected HEK293 cells in the cell-attached patchconfiguration at different holding potentials (in NaCl buffer with theaddition of 0.1 mM EGTA and 5 mM menthol in the pipette). E, Singlechannel current amplitude was obtained by measuring mean currentamplitudes at various positive holding potentials, as shown. Slopeconductance was obtained by linear regression fit, yielding a singlechannel conductance of 83±3 pS (n=3).

FIG. 4A-F. The menthol receptor is cold sensitive. A, Inward currents(top) were evoked in the same menthol receptor-expressing oocyte byrepetitive decreases in perfusate temperature. Cooling ramps (bottom)were applied at two different rates (0.2° C./s; 1° C./s). B,Temperature-response profile of the cold-evoked currents shown in panel(A). C, Response profiles of cold-evoked currents in seven independentoocytes in the absence (black lines) or presence of a sub-activatingconcentration of menthol (20 μM, gray lines). D, Inward currents evokedin a menthol receptor-expressing oocyte by a saturating cold stimulus(35 to 5° C.) were smaller than those evoked by a maximal dose of roomtemperature menthol (500 μM). E, Current-voltage relationship for a cold(14° C.)-evoked stimulus in menthol receptor-transfected cells in theabsence or presence of sub-activating dose (10 μM) of menthol. Mentholinduced potentiation and outward rectification of cold-evoked currentsare also evident in the accompanying current traces (above) obtained atvarious voltage steps (−120 to 70 mV). F, Current-voltage relationshipin transfected HEK293 cells for basal current at 22° before and afterwarming to 31° C.

FIG. 5A-B. CMR1 is a member of the TRP family of ion channels. A, Thepredicted amino acid sequence (SEQ ID NO:1) determined from the CMR1cDNA. Open boxes designate predicted transmembrane domains and aminoacids encompassing the conserved TRP family motif is underlined. B,Schematic comparison of CMR1 with other TRPM family members, TRPM2 andTRPM7. Proteins are aligned using putative transmembrane domains and TRPmotif as landmarks. Numeric label is based on the TRPM7 sequence. CMR1has a significantly shorter C-terminal tail and does not contain anyconserved domains indicative of enzymatic activity associated with TRPM2(ADP ribose pyrophosphatase, Nudix motif) or TRPM7 (protein kinase).

FIG. 6A-B. CMR1 is expressed by small-diameter neurons in trigeminal anddorsal root ganglia. A, Poly A+RNA from adult rat trigeminal ganglia(TG), dorsal root ganglia (DRG), spinal cord (SC) and brain wereanalyzed by Northern blotting, revealing two predominant transcripts of˜6 and 5 kb. The blot was re-probed with a rat cyclophilin cDNA (bottom)to control for sample loading. B, Histological sections from adult rattrigeminal or dorsal root ganglia showed selective staining (brown) witha digoxygenin-labeled CMR1 probe over neurons with small-diameter (˜19micron) cell bodies (scale bar=50 micron).

FIG. 7A-B. TRP-like channels mediate thermosensation from cold to hot.a, Schematic representation of the thermal sensitivity ranges of CMR1,VR1, and VRL-1. Based on a combination of in vitro and in vivo analyses,these channels can theoretically account for thermosensation over a widerange of ambient temperatures. The ranges of each proteins' temperaturesensitivity are denoted by bars. Other molecules may contribute totemperature sensation in zones not necessarily covered by thesechannels, in particularly those of a warm (30 to 40° C.) or extremelycold (<5° C.) nature. B, Oocytes co-expressing CMR1 and VR1 demonstratethat these channels are sufficient to confer thermal responsiveness toboth cold (menthol) and heat (capsaicin) independently. Bars abovetraces indicate application of thermal or chemical stimuli (cold, 35 to8° C.; heat, 25 to 50° C.; menthol, 100 μM; capsaicin, 1 μM).

DETAILED DESCRIPTION OF THE INVENTION

Introduction

For the first time, a protein from the TRP family of channel proteins,CMR1, has been functionally identified as a protein involved inregulating cold/menthol sensation. The present invention, therefore, hasfunctionally identified CMR1 as a drug target for compounds thatregulate the cold sensation or pain sensation, particularly pain that isstimulated by cold. Such compounds can be used in various processes thatinvolve cold perception, for example, as flavor or perfume ingredientsor as ingredients in medicinal preparations.

In some applications, the compounds may be used to elicit an effect onbreathing or thirst. It has been noted that cold air blown over themouth inhibits respiration, and that menthol compounds, which create asensation of airway cooling, also decrease the activity of upperrespiratory tract muscles (Eccles, J. Pharm. Pharmacol. 46:618-30,1994). Accordingly, CMR1 agonists could be used as a cooling agent inpreparations such as lozenges, or medications, to provide a decrease inrespiratory rate, e.g., to reduce anxiety. Menthol has also beenreported to decrease the thirst response, Similarly, CMR1 modulatorsidentified using the methods described herein could also be included inbeverages, food, or medications to decrease the thirst response.

CMR1 modulators may also be used to modulate disorders associated withcold perception, such as pain. Additionally compounds that modulate CMR1activity could also be used as inhibitors of cancer cell proliferation.Expression or repression of several TRP channel genes in tumor cellsalso suggest that these proteins have effects on cell proliferation, forexample, through their ability to regulate intracellular calcium levels.Among normal tissues examined, human TRPM8, which is 92% identical tothe CMR1 protein sequence set forth in SEQ ID NO:1, was found to beexpressed in prostate epithelia, as well as exhibiting increasedexpression in a variety of tumors, including prostate, melanoma,colorectal, and breast carcinoma (Tsavaler et al., Cancer Res. 61:3760-9(2001)). This suggests that CMR1 may function as an oncogene or tumorpromoter and that compounds that modulate CMR1 and intracellular calciumlevels may be useful as agents to inhibit cancer cell proliferation.

CMR1 proteins may also be used as biosensors, i.e., molecularthermometers.

The present invention provides isolated nucleic acid and amino acidsequences encoding CMR1 and methods of production of CMR1. Structurally,the CMR1 cDNA includes an open reading frame of 3312 bp (SEQ ID NO:2)that encodes a polypeptide of 1104 amino acids in length (SEQ ID NO:1).The amino acid sequence can be aligned with a 92% sequence identity withthe human amino acid sequence of the transient receptor potential (TRP)ion channel family, TRPM8 (or trp-p8) (see, e.g., Tsavaler et al.,Cancer Res. 61:3760-3769, 2001; U.S. Pat. No. 6,194,152, and WO99/09166).

CMR1 proteins form channels that have cation channel activity; inparticular they exhibit calcium permeability. The protein has relativelyhigh permeability to calcium and little selectivity among monovalentcations. Channel activity can be effectively measured, e.g., byrecording ligand-induced changes in [Ca²⁺]_(i) and measuring calciuminflux using fluorescent Ca²⁺-indicator dyes and fluorometric imaging.

CMR1 is expressed in a number of tissues, including sensory neurons, aswell as prostate epithelia and a variety of tumors, e.g., otherepithelial tumors. Additional tissues that may express CMR1 orhomologues include the brain and regions of the brain, such as thehypothalamus, that regulate core body temperature.

Within the TRP family, TRPM2 and TRPM7 have been electrophysiologicallycharacterized and shown to behave as bifunctional proteins in whichenzymatic activities associated with their long C-terminal domains arebelieved to regulate channel opening. Specifically, TRPM2 contains aNudix motif associated with adenosine-5′-diphosphoribose (ADPR)pyrophosphatase activity and is gated by cytoplasmic ADPR andnicotinamide adenine dinucleotide (NAD) (Perraud et al., Nature411:595-9 (2001); Sano et al., Science 293:1327-30 (2001)). TRPM7contains a protein kinase domain that is required for channel activation(Runnels et al., Science 291:1043-7 (2001)). In contrast, CMR1 has asignificantly shorter C-terminal region (FIG. 5 b) and does not containany known enzymatic domains that might be associated with channelregulation.

CMR1 encodes a channel protein that is sensitive to temperatures thatencompass all of the innocuous cool (e.g., 15 to 28° C.) and part of thenoxious cold (e.g., 8 to 15° C.) range. Furthermore, CMR1 couldcontribute to depolarization of fibers at temperatures in the ultra-coldrange (below 8° C.), for example, if the channel is modified ormodulated in a manner that extends its sensitivity range in vivo.Indeed, VR1 and several other members of the TRP channel family areregulated by receptors that couple to phospholipase C (PLC). Inparticular, the thermal activation threshold for VR1 can be markedlyshifted to lower temperatures by inflammatory agents that eitheractivate PLC signaling systems (e.g. bradykinin and nerve growth factor)or modulate the channel directly (e.g. protons and lipids) (Caterina &Julius, Annu. Rev. Neurosci. 24:487-517 (2001); Chuang et al., Nature411:957-62 (2001)).

When expressed together, CMR1 and VR1 could endow a cell with distinctthermal thresholds and temperature response ranges for cold and hot,respectively (FIG. 7 b). Indeed, calcium imaging data suggests that asignificant proportion (˜50%) of CMR1-expressing small-diameter neuronsalso express VR1 and can therefore be categorized as cold andheat-responsive nociceptors. These observations provide a molecularexplanation for the paradoxical activation of low-thresholdthermoreceptors by noxious heat (Campero et al., J. Physiol. 535:855-65(2001); Dodt & Zotterman, Acta Physiol. Scand. 26:358-365 (1952)) or forthe fact that noxious cold is sometimes perceived as burning pain (Craig& Bushnell, Science 265:252-5 (1994)). The cloning of CMR1 now makes itpossible to assess the contribution of this channel and CMR1-expressingsensory neurons to the detection of cool and cold stimuli in vivo usinghistological, electrophysiological, and genetic methods.

When applied to skin or mucous membranes, menthol produces a coolingsensation, inhibits respiratory reflexes and, at high doses, elicits apungent or irritant effect that is accompanied by local vasodilation(Eccles, J. Pharm. Pharmacol. 46:618-30 (1994); Eccles, Appetite34:29-35 (2000)). Most, if not all, of these physiological actions canbe explained by excitation of sensory nerve endings within thesetissues, but CMR1 receptors elsewhere may also contribute to these orother effects of cooling compounds or cold stimuli.

The invention also provides methods of screening for modulators, e.g.,activators, inhibitors, stimulators, enhancers, etc., of CMR1 nucleicacids and proteins, using the sequences provided herein as well asvariants, and orthologs, e.g., human orthologs, thereof. Such modulatorscan affect CMR1 activity, e.g., by modulating CMR1 transcription,translation, mRNA or protein stability; by altering the interaction ofCMR1 with the plasma membrane, or other molecules; or by affecting CMR1protein activity. Compounds are screened, e.g., using high throughputscreening (HTS), to identify those compounds that can bind to and/ormodulate the activity of a CMR1 polypeptide or fragment thereof. In oneembodiment, CMR1 proteins are recombinantly expressed in cells, e.g.,human cells, and the modulation of CMR1 is assayed by using any measureof ion channel function, such as measurement of the membrane potential,or measures of changes in intracellular calcium levels. Alternatively,endogenous CMR1 in cells, e.g., human cells, can be used for the assaysof the present invention. Methods of assaying ion, e.g., cation, channelfunction include, for example, patch clamp techniques, measurement ofwhole cell currents, and fluorescent imaging techniques that useCa²⁺-sensitive fluorescent dyes such as Fura-2.

Specific regions of the CMR1 nucleotide and amino acid sequences may beused to identify polymorphic variants, interspecies homologs, andalleles of CMR1 genes. Identification can be performed by using in vitrotechniques, e.g., by using PCR under stringent or moderate hybridizationconditions, or by using the sequence information in a computer systemfor comparison with other nucleotide sequences. Sequence comparison canbe performed using any of the sequence comparison algorithms discussedherein below. Antibodies that bind specifically to CMR1 polypeptides ora conserved region thereof, can also be used to identify alleles,interspecies homologs, and polymorphic variants.

Nucleotide and amino acid sequence information for CMR1 are also used toconstruct models of CMR1 proteins. These models are subsequently used toidentify compounds that can activate or inhibit CMR1 proteins.

A CMR1 agonist identified as set forth in the current application can beused for a number of different purposes. For example, a CMR1 activatorcan be included as a flavoring or perfuming agent in foods, beverages,soaps, medicines, soaps, etc. They can also be used in medicaments toprovide a cooling or soothing sensation.

CMR1 modulators can also be used to treat diseases or conditionsassociated with CMR1 activity, such as pain. Further, the nucleic acidand protein sequences in the current application can be used to diagnosesuch diseases or conditions.

Kits are also provided for carrying out the herein-disclosed diagnosticand therapeutic methods.

Definitions

The term “cold perception” or “cold sensation” as used herein is theability to perceive or respond to cold stimuli. Such stimuli includecold or cool temperatures, e.g., temperatures less than about 30°, andnaturally occurring or synthetic compounds such as menthol (Eccles, J.Pharm. Pharmacol 46:618-630, 1994), eucalyptol, icilin (Wei & Seid, J.Pharm. Pharmacol. 35:110-112, 1983) and the like that elicit a coldsensation.

The term “pain” refers to all categories of pain, including pain that isdescribed in terms of stimulus or nerve response, e.g., somatic pain(normal nerve response to a stimulus such as cold or menthol) andneuropathic pain (abnormal response of a injured or altered sensorypathway, often without clear noxious input); pain that is categorizedtemporally, e.g., chronic pain and acute pain; pain that is categorizedin terms of its severity, e.g., mild, moderate, or severe; and pain thatis a symptom or a result of a disease state or syndrome, e.g.,inflammatory pain, cancer pain, AIDS pain, arthropathy, migraine,trigeminal neuralgia, cardiac ischaemia, and diabetic neuropathy (see,e.g., Harrison's Principles of Internal Medicine, pp. 93-98 (Wilson etal., eds., 12th ed. 1991); Williams et al., J. of Medicinal Chem.42:1481-1485 (1999), herein each incorporated by reference in theirentirety).

“Somatic” pain, as described above, refers to a normal nerve response toa stimulus, often a noxious stimulus such as injury or illness, e.g.,cold, heat, trauma, burn, infection, inflammation, or disease processsuch as cancer, and includes both cutaneous pain (e.g., skin, muscle orjoint derived) and visceral pain (e.g., organ derived).

“Neuropathic” pain, as described above, refers to pain resulting frominjury to or chronic changes in peripheral and/or central sensorypathways, where the pain often occurs or persists without an obviousnoxious input.

“Cation channels” are a diverse group of proteins that regulate the flowof cations across cellular membranes. The ability of a specific cationchannel to transport particular cations typically varies with thevalency of the cations, as well as the specificity of the given channelfor a particular cation.

“Homomeric channel” refers to a cation channel composed of identicalalpha subunits, whereas “heteromeric channel” refers to a cation channelcomposed of two or more different types of alpha subunits. Bothhomomeric and heteromeric channels can include auxiliary beta subunits.

A “beta subunit” is a polypeptide monomer that is an auxiliary subunitof a cation channel composed of alpha subunits; however, beta subunitsalone cannot form a channel (see, e.g., U.S. Pat. No. 5,776,734). Betasubunits are known, for example, to increase the number of channels byhelping the alpha subunits reach the cell surface, change activationkinetics, and change the sensitivity of natural ligands binding to thechannels. Beta subunits can be outside of the pore region and associatedwith alpha subunits comprising the pore region. They can also contributeto the external mouth of the pore region.

The terms “CMR1” protein or fragment thereof, or a nucleic acid encoding“CMR1” or a fragment thereof refer to nucleic acids and polypeptidepolymorphic variants, alleles, mutants, and interspecies homologs that:(1) have an amino acid sequence that has greater than about 60% aminoacid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequenceidentity, preferably over a region of at least about 25, 50, 100, 200,500, 1000, or more amino acids, to an amino acid sequence encoded by aCMR1 nucleic acid or amino acid sequence of a CMR1 protein, e.g., SEQ IDNO:1 or 3; (2) specifically bind to antibodies, e.g., polyclonalantibodies, raised against an immunogen comprising an amino acidsequence of a CMR1 protein or immunogenic fragments thereof, andconservatively modified variants thereof; (3) specifically hybridizeunder stringent hybridization conditions to an anti-sense strandcorresponding to a nucleic acid sequence (SEQ ID NO:2 or 4) encoding aCMR1 protein, and conservatively modified variants thereof; (4) have anucleic acid sequence that has greater than about 60% sequence identity,65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99%, or higher nucleotide sequence identity, preferably overa region of at least about 25, 50, 100, 200, 500, 1000, or morenucleotides, to a CMR1 nucleic acid, e.g., SEQ ID NO:2 or 4. The nucleicacid and amino acid sequences for rat CMR1 have been deposited underGenBank Accession No. AY072788 and NM_(—)134371, see also McKemy et al.,Nature 416:52-58 (2002) and SEQ ID NO:1 and 3 herein. The nucleic acidand amino acid sequences for human CMR1 have been deposited underGenBank Accession No. NM_(—)024080 and AY090109, see also Tsavaler etal., Cancer Res. 61:3760-3769, 2001; U.S. Pat. No. 6,194,152, and WO99/09166 and SEQ ID NO: 3 and 4 herein. The nucleic acid and amino acidsequences for mouse CMR1 have been deposited under GenBank Accession No.NM_(—)134252, see also Peier et al., Cell 108:705-715 (2002).

A CMR1 polynucleotide or polypeptide sequence is typically from a mammalincluding, but not limited to, primate, e.g., human; rodent, e.g., rat,mouse, hamster; cow, pig, horse, sheep, or any mammal. The nucleic acidsand proteins of the invention include both naturally occurring orrecombinant molecules. CMR1 proteins typically have calcium ion channelactivity, i.e., they are permeable to calcium.

By “determining the functional effect” or “determining the effect on thecell” is meant assaying the effect of a compound that increases ordecreases a parameter that is indirectly or directly under the influenceof a CMR1 polypeptide e.g., functional, physical, phenotypic, andchemical effects. Such functional effects include, but are not limitedto, changes in ion flux, membrane potential, current amplitude, andvoltage gating, a as well as other biological effects such as changes ingene expression of CMR1 or of any marker genes, and the like. The ionflux can include any ion that passes through the channel, e.g., calcium,and analogs thereof such as radioisotopes. Such functional effects canbe measured by any means known to those skilled in the art, e.g., patchclamping, using voltage-sensitive dyes, or by measuring changes inparameters such as spectroscopic characteristics (e.g., fluorescence,absorbance, refractive index), hydrodynamic (e.g., shape),chromatographic, or solubility properties,.

“Inhibitors,” “activators,” and “modulators” of CMR1 polynucleotide andpolypeptide sequences are used to refer to activating, inhibitory, ormodulating molecules identified using in vitro and in vivo assays ofCMR1 polynucleotide and polypeptide sequences. Inhibitors are compoundsthat, e.g., bind to, partially or totally block activity, decrease,prevent, delay activation, inactivate, desensitize, or down regulate theactivity or expression of CMR1 proteins, e.g., antagonists. “Activators”are compounds that increase, open, activate, facilitate, enhanceactivation, sensitize, agonize, or up regulate CMR1 protein activity.Inhibitors, activators, or modulators also include genetically modifiedversions of CMR1 proteins, e.g., versions with altered activity, as wellas naturally occurring and synthetic ligands, antagonists, agonists,peptides, cyclic peptides, nucleic acids, antibodies, antisensemolecules, siRNA, ribozymes, small organic molecules and the like. Suchassays for inhibitors and activators include, e.g., expressing CMR1protein in vitro, in cells, cell extracts, or cell membranes, applyingputative modulator compounds, and then determining the functionaleffects on activity, as described above.

Samples or assays comprising CMR1 proteins that are treated with apotential activator, inhibitor, or modulator are compared to controlsamples without the inhibitor, activator, or modulator to examine theextent of activation or migration modulation. Control samples (untreatedwith inhibitors) are assigned a relative protein activity value of 100%.Inhibition of CMR1 is achieved when the activity value relative to thecontrol is about 80%, preferably 50%, more preferably 25-0%. Activationof CMR1 is achieved when the activity value relative to the control(untreated with activators) is 110%, more preferably 150%, morepreferably 200-500% (i.e., two to five fold higher relative to thecontrol), more preferably 1000-3000% higher.

The term “test compound” or “drug candidate” or “modulator” orgrammatical equivalents as used herein describes any molecule, eithernaturally occurring or synthetic, e.g., protein, oligopeptide (e.g.,from about 5 to about 25 amino acids in length, preferably from about 10to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 aminoacids in length), small organic molecule, polysaccharide, lipid, fattyacid, polynucleotide, siRNA, oligonucleotide, ribozyme, etc., to betested for the capacity to modulate cold sensation. The test compoundcan be in the form of a library of test compounds, such as acombinatorial or randomized library that provides a sufficient range ofdiversity. Test compounds are optionally linked to a fusion partner,e.g., targeting compounds, rescue compounds, dimerization compounds,stabilizing compounds, addressable compounds, and other functionalmoieties. Conventionally, new chemical entities with useful propertiesare generated by identifying a test compound (called a “lead compound”)with some desirable property or activity, e.g., inhibiting activity,creating variants of the lead compound, and evaluating the property andactivity of those variant compounds. Often, high throughput screening(HTS) methods are employed for such an analysis.

A “small organic molecule” refers to an organic molecule, eithernaturally occurring or synthetic, that has a molecular weight of morethan about 50 daltons and less than about 2500 daltons, preferably lessthan about 2000 daltons, preferably between about 100 to about 1000daltons, more preferably between about 200 to about 500 daltons.

“Biological sample” include sections of tissues such as biopsy andautopsy samples, and frozen sections taken for histologic purposes. Suchsamples include blood, sputum, tissue, cultured cells, e.g., primarycultures, explants, and transformed cells, stool, urine, etc. Abiological sample is typically obtained from a eukaryotic organism, mostpreferably a mammal such as a primate e.g., chimpanzee or human; cow;dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird;reptile; or fish.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region (e.g., nucleotide sequences SEQ ID NO:1), when comparedand aligned for maximum correspondence over a comparison window ordesignated region) as measured using a BLAST or BLAST 2.0 sequencecomparison algorithms with default parameters described below, or bymanual alignment and visual inspection (see, e.g., NCBI web site or thelike). Such sequences are then said to be “substantially identical.”This definition also refers to, or may be applied to, the compliment ofa test sequence. The definition also includes sequences that havedeletions and/or additions, as well as those that have substitutions. Asdescribed below, the preferred algorithms can account for gaps and thelike. Preferably, identity exists over a region that is at least about25 amino acids or nucleotides in length, or more preferably over aregion that is 50-100 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information. This algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul et al., supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form, andcomplements thereof. The term encompasses nucleic acids containing knownnucleotide analogs or modified backbone residues or linkages, which aresynthetic, naturally occurring, and non-naturally occurring, which havesimilar binding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated.

Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka etal., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell.Probes 8:91-98 (1994)). The term nucleic acid is used interchangeablywith gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

A particular nucleic acid sequence also implicitly encompasses “splicevariants.” Similarly, a particular protein encoded by a nucleic acidimplicitly encompasses any protein encoded by a splice variant of thatnucleic acid. “Splice variants,” as the name suggests, are products ofalternative splicing of a gene. After transcription, an initial nucleicacid transcript may be spliced such that different (alternate) nucleicacid splice products encode different polypeptides. Mechanisms for theproduction of splice variants vary, but include alternate splicing ofexons. Alternate polypeptides derived from the same nucleic acid byread-through transcription are also encompassed by this definition. Anyproducts of a splicing reaction, including recombinant forms of thesplice products, are included in this definition. An example ofpotassium channel splice variants is discussed in Leicher, et al., J.Biol. Chem. 273(52):35095-35101 (1998).

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence withrespect to the expression product, but not with respect to actual probesequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3^(rd) ed., 1994) and Cantor and Schimmel,Biophysical Chemistry Part I: The Conformation of BiologicalMacromolecules (1980). “Primary structure” refers to the amino acidsequence of a particular peptide. “Secondary structure” refers tolocally ordered, three dimensional structures within a polypeptide.These structures are commonly known as domains, e.g., transmembranedomains, pore domains, and cytoplasmic tail domains. Domains areportions of a polypeptide that form a compact unit of the polypeptideand are typically 15 to 350 amino acids long. Exemplary domains includeextracellular domains, transmembrane domains, and cytoplasmic domains.Typical domains are made up of sections of lesser organization such asstretches of β-sheet and α-helices. “Tertiary structure” refers to thecomplete three dimensional structure of a polypeptide monomer.“Quaternary structure” refers to the three dimensional structure formedby the noncovalent association of independent tertiary units.Anisotropic terms are also known as energy terms.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins whichcan be made detectable, e.g., by incorporating a radiolabel into thepeptide or used to detect antibodies specifically reactive with thepeptide.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acids, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, preferably 10 times backgroundhybridization. Exemplary stringent hybridization conditions can be asfollowing: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or,5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDSat 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous reference, e.g., andCurrent Protocols in Molecular Biology, ed. Ausubel, et al.

For PCR, a temperature of about 36° C. is typical for low stringencyamplification, although annealing temperatures may vary between about32° C. and 48° C. depending on primer length. For high stringency PCRamplification, a temperature of about 62° C. is typical, although highstringency annealing temperatures can range from about 50° C. to about65° C., depending on the primer length and specificity. Typical cycleconditions for both high and low stringency amplifications include adenaturation phase of 90° C.-95° C. for 30 sec-2 min., an annealingphase lasting 30 sec.-2 min., and an extension phase of about 72° C. for1-2 min. Protocols and guidelines for low and high stringencyamplification reactions are provided, e.g., in Innis et al. (1990) PCRProtocols, A Guide to Methods and Applications, Academic Press, Inc.N.Y.).

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.Typically, the antigen-binding region of an antibody will be mostcritical in specificity and affinity of binding.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990))

For preparation of antibodies, e.g., recombinant, monoclonal, orpolyclonal antibodies, many technique known in the art can be used (see,e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan,Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, ALaboratory Manual (1988) and Harlow & Lane, Using Antibodies, ALaboratory Manual (1999); and Goding, Monoclonal Antibodies: Principlesand Practice (2d ed. 1986)). The genes encoding the heavy and lightchains of an antibody of interest can be cloned from a cell, e.g., thegenes encoding a monoclonal antibody can be cloned from a hybridoma andused to produce a recombinant monoclonal antibody. Gene librariesencoding heavy and light chains of monoclonal antibodies can also bemade from hybridoma or plasma cells. Random combinations of the heavyand light chain gene products generate a large pool of antibodies withdifferent antigenic specificity (see, e.g., Kuby, Immunology (3^(rd) ed.1997)). Techniques for the production of single chain antibodies orrecombinant antibodies (U.S. Pat. Nos. 4,946,778, 4,816,567) can beadapted to produce antibodies to polypeptides of this invention. Also,transgenic mice, or other organisms such as other mammals, may be usedto express humanized or human antibodies (see, e.g., U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, Markset al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al.,Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology14:826 (1996); and Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93(1995)). Alternatively, phage display technology can be used to identifyantibodies and heteromeric Fab fragments that specifically bind toselected antigens (see, e.g., McCafferty et al., Nature 348:552-554(1990); Marks et al., Biotechnology 10:779-783 (1992)). Antibodies canalso be made bispecific, i.e., able to recognize two different antigens(see, e.g., WO 93/08829, Traunecker et al., EMBO J. 10:3655-3659 (1991);and Suresh et al., Methods in Enzymology 121:210 (1986)). Antibodies canalso be heteroconjugates, e.g., two covalently joined antibodies, orimmunotoxins (see, e.g., U.S. Pat. No. 4,676,980, WO 91/00360; WO92/200373; and EP 03089).

Methods for humanizing or primatizing non-human antibodies are wellknown in the art. Generally, a humanized antibody has one or more aminoacid residues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as import residues,which are typically taken from an import variable domain. Humanizationcan be essentially performed following the method of Winter andco-workers (see, e.g., Jones et al., Nature 321:522-525 (1986);Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science239:1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596(1992)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. Accordingly, such humanizedantibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies.

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

In one embodiment, the antibody is conjugated to an “effector” moiety.The effector moiety can be any number of molecules, including labelingmoieties such as radioactive labels or fluorescent labels, or can be atherapeutic moiety. In one aspect the antibody modulates the activity ofthe protein.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein, often in a heterogeneous population ofproteins and other biologics. Thus, under designated immunoassayconditions, the specified antibodies bind to a particular protein atleast two times the background and more typically more than 10 to 100times background. Specific binding to an antibody under such conditionsrequires an antibody that is selected for its specificity for aparticular protein. For example, polyclonal antibodies raised to CMR1protein as encoded by SEQ ID NO:1, polymorphic variants, alleles,orthologs, and conservatively modified variants, or splice variants, orportions thereof, can be selected to obtain only those polyclonalantibodies that are specifically immunoreactive with CMR1 proteins andnot with other proteins. This selection may be achieved by subtractingout antibodies that cross-react with other molecules. A variety ofimmunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select antibodies specificallyimmunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, ALaboratory Manual (1988) for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity).

By “therapeutically effective dose” herein is meant a dose that produceseffects for which it is administered. The exact dose will depend on thepurpose of the treatment, and will be ascertainable by one skilled inthe art using known techniques (see, e.g., Lieberman, PharmaceuticalDosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technologyof Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations(1999)).

Isolation of Nucleic Acids Encoding CMR1 Proteins

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook and Russell, Molecular Cloning, A LaboratoryManual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

CMR1 nucleic acids, polymorphic variants, orthologs, and alleles thatare substantially identical to an amino acid sequence encoded by SEQ IDNO:1, as well as other CMR1 family members, can be isolated using CMR1nucleic acid probes and oligonucleotides under stringent hybridizationconditions, by screening libraries. Alternatively, expression librariescan be used to clone CMR1 protein, polymorphic variants, orthologs, andalleles by detecting expressed homologs immunologically with antisera orpurified antibodies made against human CMR1 or portions thereof.

To make a cDNA library, one should choose a source that is rich in CMR1RNA. The mRNA is then made into cDNA using reverse transcriptase,ligated into a recombinant vector, and transfected into a recombinanthost for propagation, screening and cloning. Methods for making andscreening cDNA libraries are well known (see, e.g., Gubler & Hoffman,Gene 25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra).

For a genomic library, the DNA is extracted from the tissue and eithermechanically sheared or enzymatically digested to yield fragments ofabout 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in bacteriophagelambda vectors. These vectors and phage are packaged in vitro.Recombinant phage are analyzed by plaque hybridization as described inBenton & Davis, Science 196:180-182 (1977). Colony hybridization iscarried out as generally described in Grunstein et al., Proc. Natl.Acad. Sci. USA., 72:3961-3965 (1975).

An alternative method of isolating CMR1 nucleic acid and its orthologs,alleles, mutants, polymorphic variants, and conservatively modifiedvariants combines the use of synthetic oligonucleotide primers andamplification of an RNA or DNA template (see U.S. Pat. Nos. 4,683,195and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Inniset al., eds, 1990)). Methods such as polymerase chain reaction (PCR) andligase chain reaction (LCR) can be used to amplify nucleic acidsequences of human CMR1 directly from mRNA, from cDNA, from genomiclibraries or cDNA libraries. Degenerate oligonucleotides can be designedto amplify CMR1 homologs using the sequences provided herein.Restriction endonuclease sites can be incorporated into the primers.Polymerase chain reaction or other in vitro amplification methods mayalso be useful, for example, to clone nucleic acid sequences that codefor proteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of CMR1 encoding mRNA in physiological samples,for nucleic acid sequencing, or for other purposes. Genes amplified bythe PCR reaction can be purified from agarose gels and cloned into anappropriate vector.

Gene expression of CMR1 can also be analyzed by techniques known in theart, e.g., reverse transcription and amplification of mRNA, isolation oftotal RNA or poly A′ RNA, northern blotting, dot blotting, in situhybridization, RNase protection, high density polynucleotide arraytechnology, e.g., and the like.

Nucleic acids encoding CMR1 protein can be used with high densityoligonucleotide array technology (e.g., GeneChip™) to identify CMR1protein, orthologs, alleles, conservatively modified variants, andpolymorphic variants in this invention. In the case where the homologsbeing identified are linked to modulation of T cell activation andmigration, they can be used with GeneChip™ as a diagnostic tool indetecting the disease in a biological sample, see, e.g., Gunthand etal., AIDS Res. Hum. Retroviruses 14: 869-876 (1998); Kozal et al., Nat.Med. 2:753-759 (1996); Matson et al., Anal. Biochem. 224:110-106 (1995);Lockhart et al., Nat. Biotechnol. 14:1675-1680 (1996); Gingeras et al.,Genome Res. 8:435-448 (1998); Hacia et al., Nucleic Acids Res.26:3865-3866 (1998).

The gene for CMR1 is typically cloned into intermediate vectors beforetransformation into prokaryotic or eukaryotic cells for replicationand/or expression. These intermediate vectors are typically prokaryotevectors, e.g., plasmids, or shuttle vectors.

Expression in Prokaryotes and Eukaryotes

To obtain high level expression of a cloned gene, such as those cDNAsencoding CMR1, one typically subclones CMR1 into an expression vectorthat contains a strong promoter to direct transcription, atranscription/translation terminator, and if for a nucleic acid encodinga protein, a ribosome binding site for translational initiation.Suitable bacterial promoters are well known in the art and described,e.g., in Sambrook et al., and Ausubel et al., supra. Bacterialexpression systems for expressing the CMR1 protein are available in,e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983). Kits forsuch expression systems are commercially available. Eukaryoticexpression systems for mammalian cells, yeast, and insect cells are wellknown in the art and are also commercially available. In one preferredembodiment, retroviral expression systems are used in the presentinvention.

Selection of the promoter used to direct expression of a heterologousnucleic acid depends on the particular application. The promoter ispreferably positioned about the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the CMR1-encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operably linked to the nucleic acid sequence encoding CMR1and signals required for efficient polyadenylation of the transcript,ribosome binding sites, and translation termination. Additional elementsof the cassette may include enhancers and, if genomic DNA is used as thestructural gene, introns with functional splice donor and acceptorsites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as MBP, GST, and LacZ. Epitope tags can also beadded to recombinant proteins to provide convenient methods ofisolation, e.g., c-myc. Sequence tags may be included in an expressioncassette for nucleic acid rescue. Markers such as fluorescent proteins,green or red fluorescent protein, β-gal, CAT, and the like can beincluded in the vectors as markers for vector transduction.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, retroviral vectors, and vectorsderived from Epstein-Barr virus. Other exemplary eukaryotic vectorsinclude pMSG, pAV009/A⁺, pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, andany other vector allowing expression of proteins under the direction ofthe CMV promoter, SV40 early promoter, SV40 later promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells.

Expression of proteins from eukaryotic vectors can be also be regulatedusing inducible promoters. With inducible promoters, expression levelsare tied to the concentration of inducing agents, such as tetracyclineor ecdysone, by the incorporation of response elements for these agentsinto the promoter. Generally, high level expression is obtained frominducible promoters only in the presence of the inducing agent; basalexpression levels are minimal.

In one embodiment, the vectors of the invention have a regulatablepromoter, e.g., tet-regulated systems and the RU-486 system (see, e.g.,Gossen & Bujard, Proc. Nat'l Acad. Sci. USA 89:5547 (1992); Oligino etal., Gene Ther. 5:491-496 (1998); Wang et al., Gene Ther. 4:432-441(1997); Neering et al., Blood 88:1147-1155 (1996); and Rendahl et al.,Nat. Biotechnol. 16:757-761 (1998)). These impart small molecule controlon the expression of the candidate target nucleic acids. This beneficialfeature can be used to determine that a desired phenotype is caused by atransfected cDNA rather than a somatic mutation.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase and dihydrofolate reductase. Alternatively,high yield expression systems not involving gene amplification are alsosuitable, such as using a baculovirus vector in insect cells, with aCMR1 encoding sequence under the direction of the polyhedrin promoter orother strong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of CMR1protein, which are then purified using standard techniques (see, e.g.,Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

Any of the well-known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,biolistics, liposomes, microinjection, plasma vectors, viral vectors andany of the other well known methods for introducing cloned genomic DNA,cDNA, synthetic DNA or other foreign genetic material into a host cell(see, e.g., Sambrook et al., supra). It is only necessary that theparticular genetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressingCMR1.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofCMR1, which is recovered from the culture using standard techniquesidentified below.

Purification of CMR1 Polypeptides

Either naturally occurring or recombinant CMR1 can be purified for usein functional assays. Naturally occurring CMR1 can be purified, e.g.,from human tissue. Recombinant CMR1 can be purified from any suitableexpression system.

The CMR1 protein may be purified to substantial purity by standardtechniques, including selective precipitation with such substances asammonium sulfate; column chromatography, immunopurification methods, andothers (see, e.g., Scopes, Protein Purification Principles and Practice(1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook etal., supra).

A number of procedures can be employed when recombinant CMR1 protein isbeing purified. For example, proteins having established molecularadhesion properties can be reversible fused to the CMR1 protein. Withthe appropriate ligand, CMR1 protein can be selectively adsorbed to apurification column and then freed from the column in a relatively pureform. The fused protein is then removed by enzymatic activity. Finally,CMR1 protein could be purified using immunoaffinity columns.

A. Purification of CMR1 from Recombinant Bacteria

Recombinant proteins are expressed by transformed bacteria in largeamounts, typically after promoter induction; but expression can beconstitutive. Promoter induction with IPTG is one example of aninducible promoter system. Bacteria are grown according to standardprocedures in the art. Fresh or frozen bacteria cells are used forisolation of protein.

Proteins expressed in bacteria may form insoluble aggregates (“inclusionbodies”). Several protocols are suitable for purification of CMR1protein inclusion bodies. For example, purification of inclusion bodiestypically involves the extraction, separation and/or purification ofinclusion bodies by disruption of bacterial cells, e.g., by incubationin a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT,0.1 mM ATP, and 1 mM PMSF. The cell suspension can be lysed using 2-3passages through a French Press, homogenized using a Polytron (BrinkmanInstruments) or sonicated on ice. Alternate methods of lysing bacteriaare apparent to those of skill in the art (see, e.g., Sambrook et al.,supra; Ausubel et al., supra).

If necessary, the inclusion bodies are solubilized, and the lysed cellsuspension is typically centrifuged to remove unwanted insoluble matter.Proteins that formed the inclusion bodies may be renatured by dilutionor dialysis with a compatible buffer. Suitable solvents include, but arenot limited to urea (from about 4 M to about 8 M), formamide (at leastabout 80%, volume/volume basis), and guanidine hydrochloride (from about4 M to about 8 M). Some solvents which are capable of solubilizingaggregate-forming proteins, for example SDS (sodium dodecyl sulfate),70% formic acid, are inappropriate for use in this procedure due to thepossibility of irreversible denaturation of the proteins, accompanied bya lack of immunogenicity and/or activity. Although guanidinehydrochloride and similar agents are denaturants, this denaturation isnot irreversible and renaturation may occur upon removal (by dialysis,for example) or dilution of the denaturant, allowing re-formation ofimmunologically and/or biologically active protein. Other suitablebuffers are known to those skilled in the art. Human CMR1 proteins areseparated from other bacterial proteins by standard separationtechniques, e.g., with Ni-NTA agarose resin.

Alternatively, it is possible to purify CMR1 protein from bacteriaperiplasm. After lysis of the bacteria, when the CMR1 protein exportedinto the periplasm of the bacteria, the periplasmic fraction of thebacteria can be isolated by cold osmotic shock in addition to othermethods known to skill in the art. To isolate recombinant proteins fromthe periplasm, the bacterial cells are centrifuged to form a pellet. Thepellet is resuspended in a buffer containing 20% sucrose. To lyse thecells, the bacteria are centrifuged and the pellet is resuspended inice-cold 5 mM MgSO₄ and kept in an ice bath for approximately 10minutes. The cell suspension is centrifuged and the supernatant decantedand saved. The recombinant proteins present in the supernatant can beseparated from the host proteins by standard separation techniques wellknown to those of skill in the art.

B. Standard Protein Separation Techniques for Purifying CMR1 Proteins

Solubility Fractionation

Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

Size Differential Filtration

The molecular weight of the CMR1 proteins can be used to isolate it fromproteins of greater and lesser size using ultrafiltration throughmembranes of different pore size (for example, Amicon or Milliporemembranes). As a first step, the protein mixture is ultrafilteredthrough a membrane with a pore size that has a lower molecular weightcut-off than the molecular weight of the protein of interest. Theretentate of the ultrafiltration is then ultrafiltered against amembrane with a molecular cut off greater than the molecular weight ofthe protein of interest. The recombinant protein will pass through themembrane into the filtrate. The filtrate can then be chromatographed asdescribed below.

Column Chromatography

The CMR1 proteins can also be separated from other proteins on the basisof its size, net surface charge, hydrophobicity, and affinity forligands. In addition, antibodies raised against proteins can beconjugated to column matrices and the proteins immunopurified. All ofthese methods are well known in the art. It will be apparent to one ofskill that chromatographic techniques can be performed at any scale andusing equipment from many different manufacturers (e.g., PharmaciaBiotech).

Assays For Modulators Of CMR1 Protein

A. Assays

Modulation of a CMR1 protein, and corresponding modulation of lymphocyteactivation and/or migration, can be assessed using a variety of in vitroand in vivo assays, including cell-based models as described above. Suchassays can be used to test for inhibitors and activators of CMR1 proteinor fragments thereof, and, consequently, inhibitors and activators ofcold sensation. Such modulators of CMR1 protein are useful for creatinga perception of coolness, e.g., for use in medications or as flavorings,or treating disorders related to cold perception. Modulators of CMR1protein are tested using either recombinant or naturally occurring CMR1.

Often, the CMR1 protein will have the sequence as encoded by SEQ IDNO:1, or a human ortholog such as TRPM-8 (e.g., U.S. Pat. No. 6,194,152and WO 99/09166) or a conservatively modified variant thereof.Alternatively, the CMR1 protein of the assay will be derived from aeukaryote and include an amino acid subsequence having substantial aminoacid sequence identity to SEQ ID NO:1. Generally, the amino acidsequence identity will be at least 60%, preferably at least 65%, 70%,75%, 80%, 85%, or 90%, most preferably at least 95%, e.g., 96%, 97%, 98%or 99%.

Measurement of cold sensation phenotype of CMR1 protein or cellexpressing CMR1 protein, either recombinant or naturally occurring, canbe performed using a variety of assays, in vitro, in vivo, and ex vivo,as described herein. To identify molecules capable of modulating CMR1,assays are performed to detect the effect of various candidatemodulators on CMR1 activity in a cell.

The channel activity of CMR1 proteins can be assayed using a variety ofassays to measure changes in ion fluxes including patch clamptechniques, measurement of whole cell currents, radiolabeled ion fluxassays, and fluorescence assays using voltage-sensitive dyes (see, e.g.,Vestergarrd-Bogind et al., J. Membrane Biol. 88:67-75 (1988); Daniel etal., J. Pharmacol. Meth. 25:185-193 (1991); Hoevinsky et al., J.Membrane Biol. 137:59-70 (1994)). For example, a nucleic acid encoding aCMR1 protein or homolog thereof can be injected into Xenopus oocytes.Channel activity can then be assessed by measuring changes in membranepolarization, i.e., changes in membrane potential. One means to obtainelectrophysiological measurements is by measuring currents using patchclamp techniques, e.g., the “cell-attached” mode, the “inside-out” mode,and the “whole cell” mode (see, e.g., Ackerman et al., New Engl. J. Med.336:1575-1595, 1997). Whole cell currents can be determined usingstandard methodology such as that described by Hamil et al., PFlugers.Archiv. 391:185 (1981).

Channel activity is also conveniently assessed by measuring changes inintracellular Ca²⁺ levels. Such methods are well known in the art. Forexample, calcium flux can be measured by assessment of the uptake of⁴⁵Ca²⁺ or by using fluorescent dyes such as Fura-2. In a typicalmicrofluorimetry assay, a dye such as Fura-2, which undergoes a changein fluorescence upon binding a single Ca²⁺ ion, is loaded into thecytosol of CMR-1-expressing cells. Upon exposure to CMR1 agonist, anincrease in cytosolic calcium is reflected by a change in fluorescenceof Fura-2 that occurs when calcium is bound.

The activity of CMR1 polypeptides can be also assessed using a varietyof other in vitro and in vivo assays to determine functional, chemical,and physical effects, e.g., measuring the binding of CMR1 to othermolecules, including peptides, small organic molecules, and lipids;measuring CMR1 protein and/or RNA levels, or measuring other aspects ofCMR1 polypeptides, e.g., transcription levels, or physiological changesthat affects CMR1 activity. When the functional consequences aredetermined using intact cells or animals, one can also measure a varietyof effects such as changes in cell growth or pH changes or changes inintracellular second messengers such as IP3, cGMP, or cAMP, orcomponents or regulators of the phospholipase C signaling pathway. Suchassays can be used to test for both activators and inhibitors of KCNBproteins. Modulators thus identified are useful for, e.g., manydiagnostic and therapeutic applications.

In vitro assays

Assays to identify compounds with CMR1 modulating activity can beperformed in vitro. Such assays can use full length CMR1 protein or avariant thereof (see, e.g., SEQ ID NO:1), or a fragment of a CMR1protein, such as an extracellular domain or a cytoplasmic domain,optionally fused to a heterologous protein to form a chimera. In oneembodiment, the full-length polypeptide can be used in high throughputbinding assays to identify compounds that modulate cold sensation.Purified recombinant or naturally occurring CMR1 protein can be used inthe in vitro methods of the invention. In addition to purified CMR1protein or fragment thereof, the recombinant or naturally occurring CMR1protein can be part of a cellular lysate or a cell membrane. Asdescribed below, the binding assay can be either solid state or soluble.Preferably, the protein, fragment thereof or membrane is bound to asolid support, either covalently or non-covalently. Often, the in vitroassays of the invention are ligand binding or ligand affinity assays,either non-competitive or competitive (with known extracellular ligandssuch as menthol). Other in vitro assays include measuring changes inspectroscopic (e.g., fluorescence, absorbance, refractive index),hydrodynamic (e.g., shape), chromatographic, or solubility propertiesfor the protein.

In one embodiment, a high throughput binding assay is performed in whichthe CMR1 protein or fragment thereof is contacted with a potentialmodulator and incubated for a suitable amount of time. In oneembodiment, the potential modulator is bound to a solid support, and theCMR1 protein is added. In another embodiment, the CMR1 protein is boundto a solid support. A wide variety of modulators can be used, asdescribed below, including small organic molecules, peptides,antibodies, and CMR1 ligand analogs. A wide variety of assays can beused to identify CMR1-modulator binding, including labeledprotein-protein binding assays, electrophoretic mobility shifts,immunoassays, enzymatic assays such as phosphorylation assays, and thelike. In some cases, the binding of the candidate modulator isdetermined through the use of competitive binding assays, whereinterference with binding of a known ligand is measured in the presenceof a potential modulator. Ligands for the CMR1 family are known (e.g.,menthol). Either the modulator or the known ligand is bound first, andthen the competitor is added. After the CMR1 protein is washed,interference with binding, either of the potential modulator or of theknown ligand, is determined. Often, either the potential modulator orthe known ligand is labeled.

High throughput functional genomics assays can also be used to identifymodulators of cold sensation by identifying compounds that disruptprotein interactions between CMR1 and other proteins to which it binds.Such assays can, e.g., monitor changes in cell surface markerexpression, changes in intracellular calcium, or changes in membranecurrents using either cell lines or primary cells. Typically, the cellsare contacted with a cDNA or a random peptide library (encoded bynucleic acids). The cDNA library can comprise sense, antisense, fulllength, and truncated cDNAs. The peptide library is encoded by nucleicacids. The effect of the cDNA or peptide library on the phenotype of thecells is then monitored, using an assay as described above. The effectof the cDNA or peptide can be validated and distinguished from somaticmutations, using, e.g., regulatable expression of the nucleic acid suchas expression from a tetracycline promoter. cDNAs and nucleic acidsencoding peptides can be rescued using techniques known to those ofskill in the art, e.g., using a sequence tag.

Proteins interacting with the peptide or with the protein encoded by thecDNA (e.g., CMR1) can be isolated using a yeast two-hybrid system,mammalian two hybrid system, or phage display screen, etc. Targets soidentified can be further used as bait in these assays to identifyadditional components that may interact with the CMR1 channel whichmembers are also targets for drug development (see, e.g., Fields et al.,Nature 340:245 (1989); Vasavada et al., Proc. Nat'l Acad. Sci. USA88:10686 (1991); Fearon et al., Proc. Nat'l Acad. Sci. USA 89:7958(1992); Dang et al., Mol. Cell. Biol. 11:954 (1991); Chien et al., Proc.Nat'l Acad. Sci. USA 9578 (1991); and U.S. Pat. Nos. 5,283,173,5,667,973, 5,468,614, 5,525,490, and 5,637,463).

Cell-based in vivo Assays

In another embodiment, CMR1 protein is expressed in a cell, andfunctional, e.g., physical and chemical or phenotypic, changes areassayed to identify CMR1 modulators that modulate cold sensations. Cellsexpressing CMR1 proteins can also be used in binding assays. Anysuitable functional effect can be measured, as described herein. Forexample, changes in membrane potential, changes in intracellular Ca²⁺levels, and ligand binding are all suitable assays to identify potentialmodulators using a cell based system. Suitable cells for such cell basedassays include both primary cells, e.g., sensory neurons from the dorsalroot ganglion and cell lines that express a CMR1 protein. The CMR1protein can be naturally occurring or recombinant. Also, as describedabove, fragments of CMR1 proteins or chimeras with ion channel activitycan be used in cell based assays. For example, a transmembrane domain ofa CMR1 protein can be fused to a cytoplasmic domain of a heterologousprotein, preferably a heterologous ion channel protein. Such a chimericprotein would have ion channel activity and could be used in cell basedassays of the invention. In another embodiment, a domain of the CMR1protein, such as the extracellular or cytoplasmic domain, is used in thecell-based assays of the invention.

In another embodiment, cellular CMR1 polypeptide levels are determinedby measuring the level of protein or mRNA. The level of CMR1 protein orproteins related to CMR1 ion channel activation are measured usingimmunoassays such as western blotting, ELISA and the like with anantibody that selectively binds to the CMR1 polypeptide or a fragmentthereof. For measurement of mRNA, amplification, e.g., using PCR, LCR,or hybridization assays, e.g., northern hybridization, RNAse protection,dot blotting, are preferred. The level of protein or mRNA is detectedusing directly or indirectly labeled detection agents, e.g.,fluorescently or radioactively labeled nucleic acids, radioactively orenzymatically labeled antibodies, and the like, as described herein.

Alternatively, CMR1 expression can be measured using a reporter genesystem. Such a system can be devised using a CMR1 protein promoteroperably linked to a reporter gene such as chloramphenicolacetyltransferase, firefly luciferase, bacterial luciferase,β-galactosidase and alkaline phosphatase. Furthermore, the protein ofinterest can be used as an indirect reporter via attachment to a secondreporter such as red or green fluorescent protein (see, e.g., Mistili &Spector, Nature Biotechnology 15:961-964 (1997)). The reporter constructis typically transfected into a cell. After treatment with a potentialmodulator, the amount of reporter gene transcription, translation, oractivity is measured according to standard techniques known to those ofskill in the art.

In another embodiment, a functional effect related to signaltransduction can be measured. An activated or inhibited CMR1 will alterthe properties of target enzymes, second messengers, channels, and othereffector proteins. The examples include the activation of phospholipaseC and other signaling systems. Downstream consequences can also beexamined such as generation of diacyl glycerol and IP3 by phospholipaseC.

Assays for CMR1 activity include cells that are loaded with ion orvoltage sensitive dyes to report receptor activity, e.g., by observingcalcium influx or intracellular calcium release. Assays for determiningactivity of such receptors can also use known agonists and antagonistsfor CMR1 receptors as negative or positive controls to assess activityof tested compounds. In assays for identifying modulatory compounds(e.g., agonists, antagonists), changes in the level of ions in thecytoplasm or membrane voltage will be monitored using an ion sensitiveor membrane voltage fluorescent indicator, respectively. Among theion-sensitive indicators and voltage probes that may be employed arethose disclosed in the Molecular Probes 1997 Catalog.

Animal Models

Animal models of cold sensation also find use in screening formodulators of lymphocyte activation or migration. Similarly, transgenicanimal technology including gene knockout technology, for example as aresult of homologous recombination with an appropriate gene targetingvector, or gene overexpression, will result in the absence or increasedexpression of the CMR1 protein. The same technology can also be appliedto make knock-out cells. When desired, tissue-specific expression orknockout of the CMR1 protein may be necessary. Transgenic animalsgenerated by such methods find use as animal models of cold responses.

Knock-out cells and transgenic mice can be made by insertion of a markergene or other heterologous gene into an endogenous CMR1 gene site in themouse genome via homologous recombination. Such mice can also be made bysubstituting an endogenous CMR1 with a mutated version of the CMR1 gene,or by mutating an endogenous CMR1, e.g., by exposure to known mutagens.

A DNA construct is introduced into the nuclei of embryonic stem cells.Cells containing the newly engineered genetic lesion are injected into ahost mouse embryo, which is re-implanted into a recipient female. Someof these embryos develop into chimeric mice that possess germ cellspartially derived from the mutant cell line. Therefore, by breeding thechimeric mice it is possible to obtain a new line of mice containing theintroduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288(1989)). Chimeric targeted mice can be derived according to Hogan etal., Manipulating the Mouse Embryo: A Laboratory Manual (1988) andTeratocarcinomas and Embryonic Stem Cells: A Practical Approach(Robertson, ed., 1987).

B. Modulators

The compounds tested as modulators of CMR1 protein can be any smallorganic molecule, or a biological entity, such as a protein, e.g., anantibody or peptide, a sugar, a nucleic acid, e.g., an antisenseoligonucleotide or a ribozyme, or a lipid. Alternatively, modulators canbe genetically altered versions of an CMR1 protein. Typically, testcompounds will be small organic molecules, peptides, lipids, and lipidanalogs. In one embodiment, the compound is a menthol analog, eithernaturally occurring or synthetic.

Essentially any chemical compound can be used as a potential modulatoror ligand in the assays of the invention, although most often compoundscan be dissolved in aqueous or organic (especially DMSO-based) solutionsare used. The assays are designed to screen large chemical libraries byautomating the assay steps and providing compounds from any convenientsource to assays, which are typically run in parallel (e.g., inmicrotiter formats on microtiter plates in robotic assays). It will beappreciated that there are many suppliers of chemical compounds,including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.),Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika(Buchs Switzerland) and the like.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial small organic molecule or peptide librarycontaining a large number of potential therapeutic compounds (potentialmodulator or ligand compounds). Such “combinatorial chemical libraries”or “ligand libraries” are then screened in one or more assays, asdescribed herein, to identify those library members (particular chemicalspecies or subclasses) that display a desired characteristic activity.The compounds thus identified can serve as conventional “lead compounds”or can themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT PublicationNo. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomerssuch as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN,January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, 5,288,514, and thelike).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc.,St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton,Pa., Martek Biosciences, Columbia, Md.).

C. Solid State and Soluble High Throughput Assays

In one embodiment the invention provides soluble assays using a CMR1protein, or a cell or tissue expressing a CMR1 protein, either naturallyoccurring or recombinant. In another embodiment, the invention providessolid phase based in vitro assays in a high throughput format, where theCMR1 protein or fragment thereof, such as the cytoplasmic domain, isattached to a solid phase substrate. Any one of the assays describedherein can be adapted for high throughput screening, e.g., ligandbinding, calcium flux, change in membrane potential, etc.

In the high throughput assays of the invention, either soluble or solidstate, it is possible to screen up to several thousand differentmodulators or ligands in a single day. This methodology can be used forCMR1 proteins in vitro, or for cell-based or membrane-based assayscomprising an CMR1 protein. In particular, each well of a microtiterplate can be used to run a separate assay against a selected potentialmodulator, or, if concentration or incubation time effects are to beobserved, every 5-10 wells can test a single modulator. Thus, a singlestandard microtiter plate can assay about 100 (e.g., 96) modulators. If1536 well plates are used, then a single plate can easily assay fromabout 100- about 1500 different compounds. It is possible to assay manyplates per day; assay screens for up to about 6,000, 20,000, 50,000, ormore than 100,000 different compounds are possible using the integratedsystems of the invention.

For a solid state reaction, the protein of interest or a fragmentthereof, e.g., an extracellular domain, or a cell or membrane comprisingthe protein of interest or a fragment thereof as part of a fusionprotein can be bound to the solid state component, directly orindirectly, via covalent or non covalent linkage e.g., via a tag. Thetag can be any of a variety of components. In general, a molecule whichbinds the tag (a tag binder) is fixed to a solid support, and the taggedmolecule of interest is attached to the solid support by interaction ofthe tag and the tag binder.

A number of tags and tag binders can be used, based upon known molecularinteractions well described in the literature. For example, where a taghas a natural binder, for example, biotin, protein A, or protein G, itcan be used in conjunction with appropriate tag binders (avidin,streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.)Antibodies to molecules with natural binders such as biotin are alsowidely available and appropriate tag binders; see, SIGMA Immunochemicals1998 catalogue SIGMA, St. Louis Mo.).

Similarly, any haptenic or antigenic compound can be used in combinationwith an appropriate antibody to form a tag/tag binder pair. Thousands ofspecific antibodies are commercially available and many additionalantibodies are described in the literature. For example, in one commonconfiguration, the tag is a first antibody and the tag binder is asecond antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs. For example, agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherein family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g. which mediate the effects of various smallligands, including steroids, thyroid hormone, retinoids and vitamin D;peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclicpolymer configurations), oligosaccharides, proteins, phospholipids andantibodies can all interact with various cell receptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, and polyacetates can also form an appropriatetag or tag binder. Many other tag/tag binder pairs are also useful inassay systems described herein, as would be apparent to one of skillupon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serveas tags, and include polypeptide sequences, such as poly Gly sequencesof between about 5 and 200 amino acids (SEQ ID NO:5). Such flexiblelinkers are known to persons of skill in the art. For example,poly(ethylene glycol) linkers are available from Shearwater Polymers,Inc. Huntsville, Ala. These linkers optionally have amide linkages,sulfhydryl linkages, or heterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety ofmethods currently available. Solid substrates are commonly derivatizedor functionalized by exposing all or a portion of the substrate to achemical reagent which fixes a chemical group to the surface which isreactive with a portion of the tag binder. For example, groups which aresuitable for attachment to a longer chain portion would include amines,hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes andhydroxyalkylsilanes can be used to functionalize a variety of surfaces,such as glass surfaces. The construction of such solid phase biopolymerarrays is well described in the literature. See, e.g., Merrifield, J.Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987)(describing synthesis of solid phase components on pins); Frank &Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of variouspeptide sequences on cellulose disks); Fodor et al., Science,251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719(1993); and Kozal et al., Nature Medicine 2(7):753759 (1996) (alldescribing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

Immunological Detection of CMR1 Polypeptides

In addition to the detection of CMR1 gene and gene expression usingnucleic acid hybridization technology, one can also use immunoassays todetect CMR1 proteins of the invention. Such assays are useful forscreening for modulators of CMR1 and lymphocyte activation andmigration, as well as for therapeutic and diagnostic applications.Immunoassays can be used to qualitatively or quantitatively analyze CMR1protein. A general overview of the applicable technology can be found inHarlow & Lane, Antibodies: A Laboratory Manual (1988).

A. Production of Antibodies

Methods of producing polyclonal and monoclonal antibodies that reactspecifically with the CMR1 proteins are known to those of skill in theart (see, e.g., Coligan, Current Protocols in Immunology (1991); Harlow& Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice(2d ed. 1986); and Kohler & Milstein, Nature 256:495-497 (1975). Suchtechniques include antibody preparation by selection of antibodies fromlibraries of recombinant antibodies in phage or similar vectors, as wellas preparation of polyclonal and monoclonal antibodies by immunizingrabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989);Ward et al., Nature 341:544-546 (1989)).

A number of immunogens comprising portions of CMR1 protein may be usedto produce antibodies specifically reactive with CMR1 protein. Forexample, recombinant CMR1 protein or an antigenic fragment thereof, canbe isolated as described herein. Recombinant protein can be expressed ineukaryotic or prokaryotic cells as described above, and purified asgenerally described above. Recombinant protein is the preferredimmunogen for the production of monoclonal or polyclonal antibodies.Alternatively, a synthetic peptide derived from the sequences disclosedherein and conjugated to a carrier protein can be used an immunogen.Naturally occurring protein may also be used either in pure or impureform. The product is then injected into an animal capable of producingantibodies. Either monoclonal or polyclonal antibodies may be generated,for subsequent use in immunoassays to measure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein using a standard adjuvant, such asFreund's adjuvant, and a standard immunization protocol. The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to the beta subunits.When appropriately high titers of antibody to the immunogen areobtained, blood is collected from the animal and antisera are prepared.Further fractionation of the antisera to enrich for antibodies reactiveto the protein can be done if desired (see, Harlow & Lane, supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see, Kohler & Milstein, Eur. J. Immunol. 6:511-519(1976)). Alternative methods of immortalization include transformationwith Epstein Barr Virus, oncogenes, or retroviruses, or other methodswell known in the art. Colonies arising from single immortalized cellsare screened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse, etal., Science 246:1275-1281 (1989).

Monoclonal antibodies and polyclonal sera are collected and titeredagainst the immunogen protein in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Typically, polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against non-CMR1proteins, using a competitive binding immunoassay. Specific polyclonalantisera and monoclonal antibodies will usually bind with a K_(d) of atleast about 0.1 mM, more usually at least about 1 μM, preferably atleast about 0.1 μM or better, and most preferably, 0.01 μM or better.Antibodies specific only for a particular CMR1 protein, such as humanCMR1, can also be made, by subtracting out other cross-reacting CMR1family members or orthologs from a species such as a non-human mammal.In this manner, antibodies that bind only to a particular CMR1 proteinor ortholog may be obtained.

Once the specific antibodies against CMR1 protein are available, theprotein can be detected by a variety of immunoassay methods. Inaddition, the antibody can be used therapeutically as a CMR1 modulators.For a review of immunological and immunoassay procedures, see Basic andClinical Immunology (Stites & Terr eds., 7^(th) ed. 1991). Moreover, theimmunoassays of the present invention can be performed in any of severalconfigurations, which are reviewed extensively in Enzyme Immunoassay(Maggio, ed., 1980); and Harlow & Lane, supra.

B. Immunological Binding Assays

CMR1 protein can be detected and/or quantified using any of a number ofwell recognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of thegeneral immunoassays, see also Methods in Cell Biology: Antibodies inCell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology(Stites & Terr, eds., 7th ed. 1991). Immunological binding assays (orimmunoassays) typically use an antibody that specifically binds to aprotein or antigen of choice (in this case the CMR1 protein or antigenicsubsequence thereof). The antibody (e.g., anti-CMR1) may be produced byany of a number of means well known to those of skill in the art and asdescribed above.

Immunoassays also often use a labeling agent to specifically bind to andlabel the complex formed by the antibody and antigen. The labeling agentmay itself be one of the moieties comprising the antibody/antigencomplex. Thus, the labeling agent may be a labeled CMR1 or a labeledanti-CMR1 antibody. Alternatively, the labeling agent may be a thirdmoiety, such a secondary antibody, that specifically binds to theantibody/CMR1 complex (a secondary antibody is typically specific toantibodies of the species from which the first antibody is derived).Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G may also be used as the labelagent. These proteins exhibit a strong non-immunogenic reactivity withimmunoglobulin constant regions from a variety of species (see, e.g.,Kronval et al., J. Immunol. 111:1401-1406 (1973); Akerstrom et al., J.Immunol. 135:2589-2542 (1985)). The labeling agent can be modified witha detectable moiety, such as biotin, to which another molecule canspecifically bind, such as streptavidin. A variety of detectablemoieties are well known to those skilled in the art.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, optionally from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,antigen, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

Non-competitive Assay Formats

Immunoassays for detecting CMR1 in samples may be either competitive ornoncompetitive. Noncompetitive immunoassays are assays in which theamount of antigen is directly measured. In one preferred “sandwich”assay, for example, the anti-CMR1 antibodies can be bound directly to asolid substrate on which they are immobilized. These immobilizedantibodies then capture CMR1 present in the test sample. CMR1 proteinsthus immobilized are then bound by a labeling agent, such as a secondCMR1 antibody bearing a label. Alternatively, the second antibody maylack a label, but it may, in turn, be bound by a labeled third antibodyspecific to antibodies of the species from which the second antibody isderived. The second or third antibody is typically modified with adetectable moiety, such as biotin, to which another moleculespecifically binds, e.g., streptavidin, to provide a detectable moiety.

Competitive Assay Formats

In competitive assays, the amount of CMR1 protein present in the sampleis measured indirectly by measuring the amount of a known, added(exogenous) CMR1 protein displaced (competed away) from an anti-CMR1antibody by the unknown CMR1 protein present in a sample. In onecompetitive assay, a known amount of CMR1 protein is added to a sampleand the sample is then contacted with an antibody that specificallybinds to CMR1 protein. The amount of exogenous CMR1 protein bound to theantibody is inversely proportional to the concentration of CMR1 proteinpresent in the sample. In a particularly preferred embodiment, theantibody is immobilized on a solid substrate. The amount of CMR1 proteinbound to the antibody may be determined either by measuring the amountof CMR1 present in CMR1 protein/antibody complex, or alternatively bymeasuring the amount of remaining uncomplexed protein. The amount ofCMR1 protein may be detected by providing a labeled CMR1 molecule.

A hapten inhibition assay is another preferred competitive assay. Inthis assay the known CMR1 protein is immobilized on a solid substrate. Aknown amount of anti-CMR1 antibody is added to the sample, and thesample is then contacted with the immobilized CMR1. The amount ofanti-CMR1 antibody bound to the known immobilized CMR1 is inverselyproportional to the amount of CMR1 protein present in the sample. Again,the amount of immobilized antibody may be detected by detecting eitherthe immobilized fraction of antibody or the fraction of the antibodythat remains in solution. Detection may be direct where the antibody islabeled or indirect by the subsequent addition of a labeled moiety thatspecifically binds to the antibody as described above.

Cross-Reactivity Determinations

Immunoassays in the competitive binding format can also be used forcrossreactivity determinations. For example, a CMR1 protein can beimmobilized to a solid support. Proteins (e.g., CMR1 and homologs) areadded to the assay that compete for binding of the antisera to theimmobilized antigen. The ability of the added proteins to compete forbinding of the antisera to the immobilized protein is compared to theability of the CMR1 protein to compete with itself. The percentcrossreactivity for the above proteins is calculated, using standardcalculations. Those antisera with less than 10% crossreactivity witheach of the added proteins listed above are selected and pooled. Thecross-reacting antibodies are optionally removed from the pooledantisera by immunoabsorption with the added considered proteins, e.g.,distantly related homologs.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps an allele or polymorphic variant of a CMR1protein, to the immunogen protein. In order to make this comparison, thetwo proteins are each assayed at a wide range of concentrations and theamount of each protein required to inhibit 50% of the binding of theantisera to the immobilized protein is determined. If the amount of thesecond protein required to inhibit 50% of binding is less than 10 timesthe amount of the CMR1 protein that is required to inhibit 50% ofbinding, then the second protein is said to specifically bind to thepolyclonal antibodies generated to CMR1 immunogen.

Other Assay Formats

Western blot (immunoblot) analysis is used to detect and quantify thepresence of CMR1 in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind CMR1. The anti-CMR1 antibodies specifically bindto the CMR1 on the solid support. These antibodies may be directlylabeled or alternatively may be subsequently detected using labeledantibodies (e.g., labeled sheep anti-mouse antibodies) that specificallybind to the anti-CMR1 antibodies.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev. 5:34-41 (1986)).

Reduction of Non-Specific Binding

One of skill in the art will appreciate that it is often desirable tominimize non-specific binding in immunoassays. Particularly, where theassay involves an antigen or antibody immobilized on a solid substrateit is desirable to minimize the amount of non-specific binding to thesubstrate. Means of reducing such non-specific binding are well known tothose of skill in the art. Typically, this technique involves coatingthe substrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin are widely used with powdered milk being most preferred.

Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., 3H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to another molecules (e.g., streptavidin)molecule, which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. The ligands and their targets can be used inany suitable combination with antibodies that recognize CMR1 protein, orsecondary antibodies that recognize anti-CMR1.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidotases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see U.S. Pat. No.4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

Cellular Transfection

The present invention provides the nucleic acids of CMR1 protein for thetransfection of cells in vitro and in vivo. These nucleic acids can beinserted into any of a number of well-known vectors for the transfectionof target cells and organisms as described below. The nucleic acids aretransfected into cells, ex vivo or in vivo, through the interaction ofthe vector and the target cell. The nucleic acid, under the control of apromoter, then expresses a CMR1 protein of the present invention,thereby mitigating the effects of absent, partial inactivation, orabnormal expression of a CMR1 gene, particularly as it relates to coldsensation. The compositions are administered to a patient in an amountsufficient to elicit a therapeutic response in the patient. An amountadequate to accomplish this is defined as “therapeutically effectivedose or amount.”

Such gene therapy procedures have been used to correct acquired andinherited genetic defects, cancer, and other diseases in a number ofcontexts. The ability to express artificial genes in humans facilitatesthe prevention and/or cure of many important human diseases, includingmany diseases which are not amenable to treatment by other therapies(for a review of gene therapy procedures, see Anderson, Science256:808-813 (1992); Nabel & Feigner, TIBTECH 11:211-217 (1993); Mitani &Caskey, TIBTECH 11:162-166 (1993); Mulligan, Science 926-932 (1993);Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992);Van Brunt, Biotechnology 6(10):1149-1154 (1998); Vigne, RestorativeNeurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, BritishMedical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topicsin Microbiology and Immunology (Doerfler & Bohm eds., 1995); and Yu etal., Gene Therapy 1:13-26 (1994)).

Pharmaceutical Compositions and Administration

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered (e.g., nucleic acid, protein,modulatory compounds or transduced cell), as well as by the particularmethod used to administer the composition. Accordingly, there are a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention (see, e.g., Remington's Pharmaceutical Sciences,17^(th) ed., 1989). Administration can be in any convenient manner,e.g., by injection, oral administration, inhalation, transdermalapplication, or rectal administration.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the packaged nucleic acidsuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Tablet forms caninclude one or more of lactose, sucrose, mannitol, sorbitol, calciumphosphates, corn starch, potato starch, microcrystalline cellulose,gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearicacid, and other excipients, colorants, fillers, binders, diluents,buffering agents, moistening agents, preservatives, flavoring agents,dyes, disintegrating agents, and pharmaceutically compatible carriers.Lozenge forms can comprise the active ingredient in a flavor, e.g.,sucrose, as well as pastilles comprising the active ingredient in aninert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels, and the like containing, in addition to the activeingredient, carriers known in the art.

The compound of choice, alone or in combination with other suitablecomponents, can be made into aerosol formulations (i.e., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, intraperitoneally,intravesically or intrathecally. Parenteral administration andintravenous administration are the preferred methods of administration.The formulations of commends can be presented in unit-dose or multi-dosesealed containers, such as ampules and vials.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced by nucleic acids for ex vivo therapy can also be administeredintravenously or parenterally as described above.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular vector employed and the condition of thepatient, as well as the body weight or surface area of the patient to betreated. The size of the dose also will be determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, or transduced cell type in aparticular patient.

In determining the effective amount of the vector to be administered inthe treatment or prophylaxis of conditions owing to diminished oraberrant expression of the CMR1 protein, the physician evaluatescirculating plasma levels of the vector, vector toxicities, progressionof the disease, and the production of anti-vector antibodies. Ingeneral, the dose equivalent of a naked nucleic acid from a vector isfrom about 1 μg to 100 μg for a typical 70 kilogram patient, and dosesof vectors which include a retroviral particle are calculated to yieldan equivalent amount of therapeutic nucleic acid.

For administration, compounds and transduced cells of the presentinvention can be administered at a rate determined by the LD-50 of theinhibitor, vector, or transduced cell type, and the side-effects of theinhibitor, vector or cell type at various concentrations, as applied tothe mass and overall health of the patient. Administration can beaccomplished via single or divided doses.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Menthol and Cold Activate an Inwardly Rectifying, Ca²⁺-permeable Channelon Trigeminal Sensory Neurons

Body surfaces that are innervated by trigeminal fibers, such as the eyeand tongue, are particularly sensitive to cold and cooling compounds(e.g., Eccles, J. Pharm. Pharmacol. 46: 618-30 (1994)). Calcium imagingand electrophysiological methods were therefore used to examineresponses of dissociated rat trigeminal neurons to menthol and cold.Indeed, these stimuli produced robust increases in intracellularfree-calcium in a relatively small sub-population of trigeminal neurons(FIG. 1 a), consistent with work from others using DRG cultures (see,e.g., Reid & Flonta, Nature 413:480 (2001); Suto & Gotoh, Neuroscience92:1131-5 (1999); Okazawa et al., Neuroreport 11:2151-5 (2000)). Mentholand cold excited a largely overlapping subset of neurons, a significantfraction of which (54.5±6.1%) were also activated by capsaicin (FIG. 1a). Sensitivity to capsaicin is considered a functional hallmark ofnociceptors (primary sensory neurons that detect noxious stimuli) andthus approximately half of the menthol/cold sensitive cells may becategorized as such.

Whole-cell patch-clamp recordings also showed that a subset oftrigeminal neurons are sensitive to both menthol and cold (FIG. 1 b).Thus exposure to these stimuli elicited rapidly developing membranecurrents that were characterized by pronounced outward rectification(i.e., responses at positive holding potentials were substantiallygreater than those at negative voltages) (FIG. 1 c). Menthol- orcold-evoked currents reversed close to 0 mV (E_(rev)=−3.56±1.90 mV and−0.82±0.25, respectively; n=5), suggesting that they result from theopening of non-selective cation channels, consistent with recentobservations of cold responses in cultured DRG neurons (Reid & Flonta,supra). Ion substitution experiments further showed that these currentsdo not discriminate among monovalent cations, but exhibit significantlyhigher permeability for calcium ions (P_(ca)/P_(Na)=3.22;P_(K)/P_(Na)=1.10; P_(Cs)/P_(K)=1.22; n=6) (FIG. 1 d). We found thesebiophysical properties to be particularly interesting because they arereminiscent of VR1 and several other members of the TRP channel family(Clapham et al., Nat. Rev. Neurosci. 2:387-96 (2001)).

In trigeminal neurons, room temperature menthol evoked responses in adose-dependent manner (FIG. 1 e) with a half-maximal effectiveconcentration (EC₅₀) of 80±2.4 mM, a potency that is within two-fold ofthat determined for DRG neurons using calcium imaging (Okazawa et al.,supra). Fitting these data with the Hill equation suggests that receptoractivation requires the binding of more than one menthol molecule(η=2.2). In addition to menthol, the mint plant synthesizes structuralanalogues that also elicit a cooling sensation, albeit with reducedpotency. One of these, menthone, elicited very small currents intrigeminal neurons compared to an equivalent dose of menthol (FIG. 1 b).Cyclohexanol, an inactive synthetic menthol analogue, had no effect.Cold also elicited membrane currents in these cells in a dose-dependentmanner, with a characteristic temperature threshold of 27.1±0.5° C.(n=4) (FIG. 1 f). As reported for DRG neurons, menthol potentiated coldresponses and shifted the thermal threshold to higher temperatures(29.6±0.3° C. at 10 μM menthol). Conversely, increasing the temperatureof perfusate (from room temperature to 30° C.) completely antagonizedcurrents evoked by 100 mM menthol (FIG. 1 b). Taken together, ourfindings and those of others demonstrate that menthol and cold activatea calcium permeable channel on DRG and trigeminal sensory neurons.Moreover, our electrophysiological data show that both stimuli activatecurrents with very similar biophysical properties, supporting the ideaof a common molecular site of action.

Fluorescently labeled antibodies were used to identify cold/mentholreceptor expressing cells in trigeminal ganglia. Cold/menthol receptorantibody and lectin IB4 specifically labeled subsets of rat trigeminalganglia neurons.

Expression Cloning of a Receptor for Menthol and Other Cooling Compounds

Our electrophysiological studies demonstrate that menthol and coldactivate native conductances with intrinsic and significant permeabilityto calcium ions. We therefore reasoned that a calcium imaging-basedscreening strategy, similar to that used for molecular identification ofthe vanilloid receptor (Caterina et al., Nature 389:816-24 (1997)) couldbe used to isolate a functional cDNA encoding a menthol orcold-sensitive receptor. In light of the higher prevalence of menthol-and cold-sensitive neurons in trigeminal ganglia (14.8% versus 7.4% forDRG, n=745 and 1425 cells, respectively), we constructed a cDNAexpression library consisting of ˜2 million independent clones from thistissue. Pools containing ˜10,000 clones were transfected into humanembryonic kidney-derived HEK293 cells, which were subsequently loadedwith the calcium-sensitive fluorescent dye, Fura-2, and microscopicallyexamined for stimulus-evoked changes in intracellular calcium. We choseroom temperature menthol (500 μM) as the agonist because of thetechnical simplicity in using a pharmacological, rather than a thermalstimulus for library screening. In this way, we identified a cDNA poolthat conferred menthol sensitivity to a small proportion (<5%) oftransfected cells. Iterative sub-division and re-screening ultimatelyyielded a single menthol-responsive clone.

As noted above, menthol is one of several naturally occurring orsynthetic cooling compounds whose relative potencies in psychophysicalor electrophysiological assays span a wide range (Eccles, J. Pharm.Pharmacol. 46:618-30 (1994)). To determine how the newly identifiedreceptor responds to these compounds, we expressed the cloned cDNA inXenopus oocytes and measured electrophysiological responses to thesecompounds using whole-cell voltage-clamp methods. Robust responses wereevoked by menthol or by the super-cooling agent AG-3-5 (icilin) (see,e.g., Wei & Seid, J. Pharm. Pharmacol. 35:110-112 (1983)) with icilinshowing nearly 200-fold greater potency than menthol (EC₅₀=0.36±0.03 μMand 66.7±3.3 μM, respectively). Icilin also activated the receptor withsignificantly greater efficacy (˜2.5-fold) compared to menthol (FIG. 2a, b). Eucalyptol, the main constituent of oil of Eucalyptus, alsoelicited membrane currents, but with very decreased potency (3.4±0.4 mM)and efficacy compared to menthol or icilin. Menthone, camphor, and theinactive menthol analogue, cyclohexanol, had little or no effect, evenwhen applied at concentrations approaching their limits of solubility inaqueous buffers (>500 μM). Finally, the vanilloid receptor agonist,capsaicin, did not elicit responses in these cells.

A more detailed biophysical analysis of the cloned receptor was carriedout in transfected HEK293 cells, where menthol or icilin producedcurrents with nearly time-independent kinetics and steep outwardrectification (FIG. 3 a). Ion substitution experiments showed that, likenative menthol-evoked responses in trigeminal neurons, these currentsshowed relatively high permeability to calcium and little selectivityamong monovalent cations (P_(Ca)/P_(Na)=3.34; P_(K)/P_(Na)=1.20;P_(Cs)/P_(K)=1.14; n=4-9) (FIG. 3 b). We also found that menthol-evokedcurrents showed significant desensitization (53.9±1.7% decrease in peakcurrent between the first and second application, n=3). Interestingly,this phenomenon was largely dependent on extracellular calcium sincelittle desensitization (9.1±7%, n=3) was observed in nominallycalcium-free bath solution (FIG. 3 c). Icilin showed even strongerdesensitization, but unlike menthol, this agonist was essentiallyinactive in the absence of extracellular calcium. Similar observationswere obtained in oocytes (not shown). When measured at the singlechannel level (cell-attached patch configuration), menthol-evokedcurrents also showed pronounced outward rectification. These responseswere characterized by brief, transient openings and had a slopeconductance of 83±3 pS at positive potentials (FIG. 3 d, e). We alsoobserved events with smaller unitary currents, which may representeither subconductance states of the channel or openings that were toobrief to be resolved in our analysis.

The Menthol Receptor is Also Activated by Cold

To determine whether the menthol receptor is also a cold sensor, wetested its thermal responsiveness in oocytes by lowering the temperatureof the perfusate from ˜35° C. to ˜5° C. This elicited a robust andrapidly activating inward current (at negative holding potentials) thatwas remarkably consistent since the rate of temperature change (rangingfrom 0.2 to 1° C./second) did not influence threshold or saturationtemperatures (FIG. 4 a, b). Moreover, cold-evoked currents were directlyproportional to temperature regardless of the direction of thetemperature change (not shown). Cold-activated currents had a thermalthreshold of 25.8±0.4° C. and saturated at 8.2±0.3° C. (n=12) (FIG. 4c). Consistent with the behavior of native cold currents, addition of asub-activating concentration of menthol (20 mM) to the perfusateincreased threshold and saturation temperature to 29.7±0.3° C. and15.6±0.4° C., respectively (n=7) (FIG. 4 c). Interestingly, we foundthat menthol is a more efficacious agonist than cold since saturatingcold-evoked currents were of smaller magnitude than those obtained witha maximal dose of room temperature menthol (67.4±1.9%, n=7) (FIG. 4 d).

We also examined cold-evoked currents in menthol receptor-expressingHEK293 cells. As observed for native cold responses (FIG. 1 c),current-voltage relationships for the cloned channel showed steepoutward rectification (FIG. 4 e) and were markedly potentiated by asub-activating dose of menthol (10 μM). Menthol increased cold-evokedcurrents in both the outward and inward direction, but the effect on theinward component was more pronounced, reminiscent of the effect ofcapsaicin on VR1. Native cold-evoked responses in sensory fibers orcultured DRG neurons show adaptation to a prolonged thermal stimuluslasting several minutes (Reid & Flonta, supra; Kenshalo & Duclaux, J.Neurophysiol. 40:319-32 (1977)). We found that receptor-transfectedcells showed small outwardly rectifying basal currents at roomtemperature (˜22° C.), but that responses to a subsequent 22° C.stimulus were markedly larger after the cell had first been warmed to31° C. (FIG. 4 f). This observation suggests that the cloned receptoralso shows adaptation to thermal challenges that can be reversed uponheating to room temperature. Desensitization to cold differed from thatobserved with menthol because it was independent of extracellularcalcium (not shown). Interestingly, VR1 shows similar behavior in thatdesensitization to chemical (capsaicin) or thermal (heat) stimuli arecalcium-dependent and -independent, respectively. Taken together, ourobservations show that the cloned receptor, which we now designatecold-menthol receptor subtype 1 (CMR1), has properties identical toendogenous cold/menthol currents observed in sensory neurons fromtrigeminal ganglia, as shown here, or from dorsal root ganglia (Reid &Flonta, supra; Suto & Gotoh, supra; Okazawa et al., supra).

CMR1 is a Member of the TRP Ion Channel Family

The CMR1 cDNA sequence includes an open reading frame of 3312 by that ispredicted to encode a protein of 1104 amino acids with a molecular massof 128 kD (FIG. 5 a). Database searches revealed significant homologybetween this deduced sequence and members of the transient receptorpotential (TRP) ion channel family. Within this family, CMR1 mostclosely resembles the subgroup of long TRP channels, so named for theircharacteristically large N- and C-terminal cytoplasmic tails compared toother TRP family members (Clapham et al., Nat. Rev. Neurosci. 2:387-96(2001); Harteneck et al., Trends Neurosci. 23:159-66 (2000)). Long TRPchannels are also termed TRPM in reference to melastatin, the foundingmember of this TRP channel subgroup initially identified in melanocytesand whose expression is downregulated in melanocystic tumors (Duncan etal., Cancer Res. 58:1515-20 (1998)). Among members of this subfamily,TRPM2 and TRPM7 have been electrophysiologically characterized and shownto behave as bifunctional proteins in which enzymatic activitiesassociated with their long C-terminal domains are believed to regulatechannel opening. Specifically, TRPM2 contains a Nudix motif associatedwith adenosine-5′-diphosphoribose (ADPR) pyrophosphatase activity and isgated by cytoplasmic ADPR and nicotinamide adenine dinucleotide (NAD)(Perraud et al., Nature 411:595-9 (2001) Sano et al., Science293:1327-30 (2001)). TRPM7 contains a protein kinase domain that isrequired for channel activation (Runnels et al., Science 291:1043-7(2001)). In contrast, CMR1 has a significantly shorter C-terminal region(FIG. 5 b) and does not contain any obvious enzymatic domains that mightbe associated with channel regulation.

CMR1 is 92% identical to human TRPM8 (or trp-p8), a recently identifiedmember of the TRPM subfamily (Tsavaler et al., Cancer Res. 61:3760-9(2001)). Among normal tissues examined, TRPM8 was found to be expressedexclusively in prostate epithelia, as well as in a variety of tumors,including prostate, melanoma, colorectal, and breast carcinoma. Thepresence of this channel in sensory neurons was not assessed and wetherefore carried out northern blot and in situ hybridization studies toexamine expression of CMR1 in rat trigeminal and dorsal root ganglia.Indeed, transcripts of ˜6 and 5 kb were detected in both neuronaltissues (FIG. 6 a). At the cellular level, CMR1 transcripts were foundin a subset of sensory neurons with small-diameter cell bodies (18.2±1.1mm and 21.6±0.5 mm in dorsal root and trigeminal ganglia, respectively)(FIG. 6 b), similar in size to VR1-expressing cells (19.2±0.3 mm)(Caterina et al., Nature 398:436-41 (1999)). Consistent with calciumimaging and patch-clamp studies, a subset of small-diameter neurons werelabeled by the CMR1 probe and expression was conspicuously absent fromthe vast majority of larger-diameter cells. CMR1 transcripts were moreprevalent in trigeminal ganglia versus dorsal root ganglia, which isalso consistent with the physiological observations using neuronalcultures. Taken together, these findings suggests that within sensoryganglia, CMR1 is expressed by a sub-population of primary afferentfibers (C & Aδ).

Thus, cooling compounds and cold are detected by the same molecularentity, CMR1, on primary afferent neurons of the somatosensory system.Moreover, thermosensation over a wide temperature range is mediated by acommon molecular mechanism that uses TRP ion channels as primarytransducers of thermal stimuli. For example, as few as three ionchannels (CMR1, VR1, and VRL-1) may provide coverage for a r wide rangeof temperatures (e.g., 8 to 28° C., >43° C., and >50° C., respectively)(FIG. 7 a).

Methods

Neuronal Cell Culture and Ca2+ Microfluorimetry

Trigeminal ganglia were removed from newborn S/D rats, placed inice-cold culture medium (MEM Eagle's with Earle's BSS supplemented with10% horse serum, vitamins, penicillin/streptomycin and L-glutamine),cleaned of connective tissue/blood vessels and diced into 4-5 pieces.Ganglia were then transferred to 0.125% collagenase P (Boehringer)solution in CMF Hank's, shaken gently at 37° C. for 20 (P0 animals) to30 min (P4 animals), pelleted, and resuspended in 0.05% STV at 37° C.for 2 min. Culture medium was added to inhibit enzymatic activity andganglia were triturated gently with a fire-polished Pasteur pipette.Neurons were enriched by density gradient centrifugation as described(see, e.g., Eckert et al., J. Neurosci. Methods 77:183-90, 1997). Cellswere resuspended in complete culture medium (MEM Eagle's/Earle's BSSwith 10% horse serum, vitamins, penicillin/streptomycin, L-glutamine and100 ng/ml NGF 7S (Invitrogen) and plated onto glass coverslips coatedwith PLO (1 mg/ml, Sigma) and laminin (5 μg/ml, Invitrogen). Cultureswere maintained at 37° C. in 5% CO2 and examined 1-2 days after plating.Ca²⁺ microfluorimetry was carried out as described (Caterina et al.,Nature 389:816-24 (1997)). For cell selection prior to patch-clampanalysis, CaCl₂ and pluronic acid were omitted from the loading buffer(nominally Ca²⁺-free CIB). Cells in the recording chamber were perfusedby gravity using the VC-6 valve control system (Warner Inst.).

Mammalian Cell Electrophysiology

Trigeminal neurons responding to 50 mM menthol with an increase inintracellular Ca²⁺ were selected for patch-clamp recordings. HEK293cells were cultured in DMEM with 10% fetal bovine serum andco-transfected (Lipofectamine 2000, Invitrogen) with 1 μg CMRi plasmidand 0.1 μg enhanced green fluorescence reporter plasmid to identifytransfected cells. Cells were plated onto PLO-coated coverslips the nextday and examined two days after transfection. Gigaseals were formed withpipettes (Garner Glass type 7052, ID 1.1, OD 1.5) having a resistance of3-5 MΩ in standard pipette solution. Whole-cell voltage clamp wasperformed at a holding potential of −60 mV with a 200 ms voltage rampfrom −120 mV to +80 mV at 3.6 Hz. Data were acquired usingPulse-Pulsefit (HEKA GmbH) software. Recordings were filtered at 5 kHzand sampled at 20 kHz. Pipette solution for neuronal experimentscontained (in mM) 140 CsCl, 1 EGTA, 0.6 MgCl₂, 10 HEPES, pH 7.4(adjusted with CsOH). Standard Ca²⁺-free bath solution for whole-cellrecordings contained (in mM) 140 NaCl, 4 KCl, 2 MgCl₂, 100 nM TTX(Sigma), 10 TRIS, pH 7.4 (adjusted with HCl) with or without 100 nMdendrotoxin I (Sigma). For ion replacement experiments, pipette solutioncontained (in mM) 140 KCl, 5 CsCl, 1 EGTA, 0.6 MgCl₂, 1 4-aminopyridine,10 HEPES, pH 7.4 (adjusted with KOH). Bath solution contained (in mM)140 NaCl, 4 KCl, 2 MgCl₂, 100 nM TTX, 100 nM dendrotoxin 1,100 nMapamine (Sigma), 100 nM carybdotoxin (Sigma), 1 μM ω-conotoxin MVIIC, 1μM GVIA (Latoxan), 10 TRIS, pH 7.4 (adjusted with HCl). For monovalentcation permeability experiments perfusate contained (in mM) 140 NaCl (or140 KCl or 140 NMDG), 100 nM TTX, 1 μM nitrendipine (Sigma), 10 HEPES,10 glucose, pH 7.4 (adjusted with NaOH, KOH or HCl, respectively). Fordivalent cation permeability experiments perfusate contained (in mM) 125NMDG, 10 CaCl₂, 100 nM TTX, 1 μM nitrendipine, 10 HEPES, 10 glucose, pH7.4 (adjusted with HCl). Liquid junction potentials (measured directlyin separate experiments) did not exceed 3 mV and thus no correction forthis offset was made. Bath solution for on-cell single channelexperiments contained (in mM): 150 NaCl, 1 MgCl₂, 10 TRIS. Pipettesolution for on-cell single channel experiments contained (in mM): 150NaCl, 1 MgCl₂, 0.1 EGTA, 10 TRIS and 5 or 25 μM menthol. Recordings wereperformed at 22° C. unless noted otherwise. Temperature ramps weregenerated by cooling or heating the perfusate in a jacketed coil(Harvard Inst.) connected to a thermostat. Temperature in the proximityof the patched cell was measured using a miniature thermocouple (typeMT-29/2, Physitemp) and sampled using an ITC-18 A/D board (Instrutech)and Pulse software. Permeability ratios for monovalent cations to Na(P_(x)/P_(Na)) were calculated according to:P_(X)/P_(Na)=exp(ΔE_(rev(Na-X))F/RT), where ΔEr_(ev(Na-x)) equals thereversal potential change, F the Faraday's constant, R the universal gasconstant and T the absolute temperature. For measurements of Ca²⁺permeability P_(Ca)/P_(Na) was calculated according to Lewis, C. A. J.Physiol. 286:417-45, 1979, (equation A6).

Expression Cloning, Northern Blot Analysis and In Situ Hybridization

Trigeminal neurons from newborn rats were dissociated and enriched asdescribed Eckert, supra. Polyadenylated RNA (˜2 μg) was isolated fromthese cells (PolyA Tract Kit, Promega) and used to construct a cDNAlibrary in pcDNA3 (Invitrogen) as described (Brake et al., Nature371:519-23 (1994)). Library subpools consisting of ˜10,000 clones weretransiently transfected into HEK293 cells by lipofection and split 24hours later into 8-well glass chamber slides coated with Matrigel(Becton-Dickinson). Responses to chemical or thermal stimuli wereassessed 6-24 hours later by Fura-2 Ca²⁺-imaging. Northern blotting andin situ hybridization histochemistry were performed as described(Caterina et al., Nature 389:816-24 (1997)) using the entire CMR1 cDNAto generate ³²P— or digoxygenin-labeled probes, respectively.

Oocyte Electrophysiology

cRNA transcripts were synthesized and injected into Xenopus laevisoocytes as described (Brake, supra). Two-electrode voltage-clamprecordings were performed 2-7 days post-injection. Dose-response curvesfor cooling compounds were performed at room temperature (22-24° C.).AG-3-5 (icilin) was provided by Dr. E. Wei, University of California,Berkeley. Temperature ramps were generated by heating (˜35° C.) orcooling (˜4° C.) the perfusate in a Harvard coil and monitoringtemperature changes with a thermister placed near the oocyte.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entirety for all purposes.

What is claimed is:
 1. A method for identifying a compound thatmodulates a cool or cold sensation in a mammalian subject, the methodcomprising: (i) contacting a cell comprising a cold- andmenthol-sensitive receptor (CMR1) polypeptide with the compound, whereinthe CMR1polypeptide comprises a sequence having at least 90% identity toSEQ ID NO:3 and forms a cation channel; and (ii) determining whethersaid compound that modulates the activity of said CMR1polypeptide basedon an effect in said cell that correlates to CMR1activity, and (iii) ifsaid compound elicits the effect in said cell, further determiningwhether said compound modulates a cool/cold sensation in a mammaliansubject, thereby identifying a compound that modulates cool orcold-sensation.
 2. The method of claim 1, wherein the effect is a changein intracellular calcium.
 3. The method of claim 2, wherein the effectis measured using a fluorescent compound.
 4. The method of claim 1,wherein the effect is a change in membrane potential.
 5. The method ofclaim 1, wherein the cell is a non-neuronal cell.
 6. The method of claim1, wherein the CMR1is recombinant.
 7. The method of claim 1, wherein theCMR1polypeptide is encoded by a nucleic acid having at least 95%identity to SEQ ID NO:
 4. 8. The method of claim 1, wherein theCMR1polypeptide is encoded by a nucleic acid having at least 98%identity to SEQ ID NO:
 4. 9. The method of claim 1, wherein theCMR1polypeptide is encoded by a nucleic acid having the sequence setforth in SEQ ID NO:
 4. 10. The method of claim 1, further comprising astep of stimulating the cell with cold temperature.
 11. The method ofclaim 1, wherein the identified compound enhances cool or coldsensation.
 12. The method of claim 1, wherein the identified compoundinhibits cool or cold sensation.
 13. The method of claim 1, wherein thecell is derived from mammalian cell lines 293 or CHO.
 14. The method ofclaim 1, wherein the CMR1polypeptide has at least 95% identity to SEQ IDNO:
 3. 15. The method of claim 1, wherein the CMR1polypeptide has atleast 98% identity to SEQ ID NO:
 3. 16. The method of claim 1, whereinthe CMR1polypeptide has the amino acid sequence set forth in SEQ ID NO:3.
 17. The method of claim 1, wherein said mammal is a human.
 18. Themethod of claim 1, wherein the cation channel responds to cool or coldtemperatures.
 19. The method of claim 1, wherein the cation channelresponds to a menthol compound.